US20170183685A1 - Ungulates with Genetically Modified Immune Systems - Google Patents

Abstract

The present invention provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which lack expression of functional endogenous immunoglobulin loci. The present invention also provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which express xenogenous, such as human, immunoglobulin loci. The present invention further provides ungulate, such as porcine genomic DNA sequence of porcine heavy and light chain immunoglobulins. Such animals, tissues, organs and cells can be used in research and medical therapy. In addition, methods are provided to prepare such animals, organs, tissues, and cells.

Description

FIELD OF THE INVENTION
• The present invention provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which lack expression of functional endogenous immunoglobulin loci. The present invention also provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which express xenogenous, such as human, immunoglobulin loci. The present invention further provides ungulate, such as porcine genomic DNA sequence of porcine heavy and light chain immunoglobulins. Such animals, tissues, organs and cells can be used in research and medical therapy. In addition, methods are provided to prepare such animals, organs, tissues, and cells.
BACKGROUND OF THE INVENTION
• An antigen is an agent or substance that can be recognized by the body as ‘foreign’. Often it is only one relatively small chemical group of a larger foreign substance which acts as the antigen, for example a component of the cell wall of a bacterium. Most antigens are proteins, though carbohydrates can act as weak antigens. Bacteria, viruses and other microorganisms commonly contain many antigens, as do pollens, dust mites, molds, foods, and other substances. The body reacts to antigens by making antibodies. Antibodies (also called immunoglobulins (Igs)) are proteins that are manufactured by cells of the immune system that bind to an antigen or foreign protein. Antibodies circulate in the serum of blood to detect foreign antigens and constitute the gamma globulin part of the blood proteins. These antibodies interact chemically with the antigen in a highly specific manner, like two pieces of a jigsaw puzzle, forming an antigen/antibody complex, or immune complex. This binding neutralizes or brings about the destruction of the antigen.
• When a vertebrate first encounters an antigen, it exhibits a primary humoral immune response. If the animal encounters the same antigen after a few days the immune response is more rapid and has a greater magnitude. The initial encounter causes specific immune cell (B-cell) clones to proliferate and differentiate. The progeny lymphocytes include not only effector cells (antibody producing cells) but also clones of memory cells, which retain the capacity to produce both effector and memory cells upon subsequent stimulation by the original antigen. The effector cells live for only a few days. The memory cells live for a lifetime and can be reactivated by a second stimulation with the same antigen. Thus, when an antigen is encountered a second time, its memory cells quickly produce effector cells which rapidly produce massive quantities of antibodies.
• By exploiting the unique ability of antibodies to interact with antigens in a highly specific manner, antibodies have been developed as molecules that can be manufactured and used for both diagnostic and therapeutic applications. Because of their unique ability to bind to antigenic epitopes, polyclonal and monoclonal antibodies can be used to identify molecules carrying that epitope or can be directed, by themselves or in conjunction with another moiety, to a specific site for diagnosis or therapy. Polyclonal and monoclonal antibodies can be generated against practically any pathogen or biological target. The term polyclonal antibody refers to immune sera that usually contain pathogen-specific antibodies of various isotypes and specificities. In contrast, monoclonal antibodies consist of a single immunoglobulin type, representing one isotype with one specificity.
• In 1890, Shibasaburo Kitazato and Emil Behring conducted the fundamental experiment that demonstrated immunity can be transmitted from one animal to another by transferring the serum from an immune animal to a non-immune animal. This landmark experiment laid the foundation for the introduction of passive immunization into clinical practice. However, wide scale serum therapy was largely abandoned in the 1940s because of the toxicity associated with the administration of heterologous sera and the introduction of effective antimicrobial chemotherapy. Currently, such polyclonal antibody therapy is indicated to treat infectious diseases in relatively few situations, such as replacement therapy in immunoglobulin-deficient patients, post-exposure prophylaxis against several viruses (e.g., rabies, measles, hepatitis A and B, varicella), and toxin neutralization (diphtheria, tetanus, and botulism). Despite the limited use of serum therapy, in the United States, more than 16 metric tons of human antibodies are required each year for intravenous antibody therapy. Comparable levels of use exist in the economies of most highly industrialized countries, and the demand can be expected to grow rapidly in developing countries. Currently, human antibody for passive immunization is obtained from the pooled serum of donors. Thus, there is an inherent limitation in the amount of human antibody available for therapeutic and prophylactic therapies.
• The use of antibodies for passive immunization against biological warfare agents represents a very promising defense strategy. The final line of defense against such agents is the immune system of the exposed individual. Current defense strategies against biological weapons include such measures as enhanced epidemiologic surveillance, vaccination, and use of antimicrobial agents. Since the potential threat of biological warfare and bioterrorism is inversely proportional to the number of immune persons in the targeted population, biological agents are potential weapons only against populations with a substantial proportion of susceptible persons.
• Vaccination can reduce the susceptibility of a population against specific threats; provided that a safe vaccine exists that can induce a protective response. Unfortunately, inducing a protective response by vaccination may take longer than the time between exposure and onset of disease. Moreover, many vaccines require multiple doses to achieve a protective immune response, which would limit their usefulness in an emergency to provide rapid prophylaxis after an attack. In addition, not all vaccine recipients mount a protective response, even after receiving the recommended immunization schedule.
• Drugs can provide protection when administered after exposure to certain agents, but none are available against many potential agents of biological warfare. Currently, no small-molecule drugs are available that prevent disease following exposure to preformed toxins. The only currently available intervention that could provide a state of immediate immunity is passive immunization with protective antibody (Arturo Casadevall “Passive Antibody Administration (Immediate Immunity) as a Specific Defense Against Biological Weapons” from Emerging Infectious Diseases, Posted Sep. 12, 2002).
• In addition to providing protective immunity, modern antibody-based therapies constitute a potentially useful option against newly emergent pathogenic bacteria, fungi, virus and parasites (A. Casadevall and M. D. Scharff, Clinical Infectious Diseases 1995; 150). Therapies of patients with malignancies and cancer (C. Botti et al, Leukemia 1997; Suppl 2:S55-59; B. Bodey, S. E. Siegel, and H. E. Kaiser, Anticancer Res 1996; 16(2):661), therapy of steroid resistant rejection of transplanted organs as well as autoimmune diseases can also be achieved through the use of monoclonal or polyclonal antibody preparations (N. Bonnefoy-Berard and J. P. Revillard, J Heart Lung Transplant 1996; 15(5): 435-442; C. Colby, et al Ann Pharmacother 1996; 30(10):1164-1174; M. J. Dugan, et al, Ann Hematol 1997; 75(1-2):41 2; W. Cendrowski, Boll Ist Sieroter Milan 1997; 58(4):339-343; L. K. Kastrukoff, et al Can J Neurol Sci 1978; 5(2):175178; J. E. Walker et al J Neurol Sci 1976; 29(2-4):303309).
• Recent advances in the technology of antibody production provide the means to generate human antibody reagents, while avoiding the toxicities associated with human serum therapy. The advantages of antibody-based therapies include versatility, low toxicity, pathogen specificity, enhancement of immune function, and favorable pharmacokinetics.
• The clinical use of monoclonal antibody therapeutics has just recently emerged. Monoclonal antibodies have now been approved as therapies in transplantation, cancer, infectious disease, cardiovascular disease and inflammation. In many more monoclonal antibodies are in late stage clinical trials to treat a broad range of disease indications. As a result, monoclonal antibodies represent one of the largest classes of drugs currently in development.
• Despite the recent popularity of monoclonal antibodies as therapeutics, there are some obstacles for their use. For example, many therapeutic applications for monoclonal antibodies require repeated administrations, especially for chronic diseases such as autoimmunity or cancer. Because mice are convenient for immunization and recognize most human antigens as foreign, monoclonal antibodies against human targets with therapeutic potential have typically been of murine origin. However, murine monoclonal antibodies have inherent disadvantages as human therapeutics. For example, they require more frequent dosing to maintain a therapeutic level of monoclonal antibodies because of a shorter circulating half-life in humans than human antibodies. More critically, repeated administration of murine immunoglobulin creates the likelihood that the human immune system will recognize the mouse protein as foreign, generating a human anti-mouse antibody response, which can cause a severe allergic reaction. This possibility of reduced efficacy and safety has lead to the development of a number of technologies for reducing the immunogenicity of murine monoclonal antibodies.
• Polyclonal antibodies are highly potent against multiple antigenic targets. They have the unique ability to target and kill a plurality of “evolving targets” linked with complex diseases. Also, of all drug classes, polyclonals have the highest probability of retaining activity in the event of antigen mutation. In addition, while monoclonals have limited therapeutic activity against infectious agents, polyclonals can both neutralize toxins and direct immune responses to eliminate pathogens, as well as biological warfare agents.
• The development of polyclonal and monoclonal antibody production platforms to meet future demand for production capacity represents a promising area that is currently the subject of much research. One especially promising strategy is the introduction of human immunoglobulin genes into mice or large domestic animals. An extension of this technology would include inactivation of their endogenous immunoglobulin genes. Large animals, such as sheep, pigs and cattle, are all currently used in the production of plasma derived products, such as hyperimmune serum and clotting factors, for human use. This would support the use of human polyclonal antibodies from such species on the grounds of safety and ethics. Each of these species naturally produces considerable quantities of antibody in both serum and milk.
Arrangement of Genes Encoding Immunoglobulins
• Antibody molecules are assembled from combinations of variable gene elements, and the possibilities resulting from combining the many variable gene elements in the germline enable the host to synthesize antibodies to an extraordinarily large number of antigens. Each antibody molecule consists of two classes of polypeptide chains, light (L) chains (that can be either kappa (κ) L-chain or lambda (λ) L-chain) and heavy (H) chains. The heavy and light chains join together to define a binding region for the epitope. A single antibody molecule has two identical copies of the L chain and two of the H chain. Each of the chains is comprised of a variable region (V) and a constant region (C). The variable region constitutes the antigen-binding site of the molecule. To achieve diverse antigen recognition, the DNA that encodes the variable region undergoes gene rearrangement. The constant region amino acid sequence is specific for a particular isotype of the antibody, as well as the host which produces the antibody, and thus does not undergo rearrangement.
• The mechanism of DNA rearrangement is similar for the variable region of both the heavy- and light-chain loci, although only one joining event is needed to generate a light-chain gene whereas two are needed to generate a complete heavy-chain gene. The most common mode of rearrangement involves the looping-out and deletion of the DNA between two gene segments. This occurs when the coding sequences of the two gene segments are in the same orientation in the DNA. A second mode of recombination can occur between two gene segments that have opposite transcriptional orientations. This mode of recombination is less common, although such rearrangements can account for up to half of all V_κ to J_κ joins; the transcriptional orientation of half of the human V_κ gene segments is opposite to that of the J_κ gene segments.
• The DNA sequence encoding a complete V region is generated by the somatic recombination of separate gene segments. The V region, or V domain, of an immunoglobulin heavy or light chain is encoded by more than one gene segment. For the light chain, the V domain is encoded by two separate DNA segments. The first segment encodes the first 95-101 amino acids of the light chain and is termed a V gene segment because it encodes most of the V domain. The second segment encodes the remainder of the V domain (up to 13 amino acids) and is termed a joining or J gene segment. The joining of a V and a J gene segment creates a continuous exon that encodes the whole of the light-chain V region. To make a complete immunoglobulin light-chain messenger RNA, the V-region exon is joined to the C-region sequence by RNA splicing after transcription.
• A heavy-chain V region is encoded in three gene segments. In addition to the V and J gene segments (denoted V_H and J_H to distinguish them from the light-chain V_L and J_L), there is a third gene segment called the diversity or D_H gene segment, which lies between the V_H and J_H gene segments. The process of recombination that generates a complete heavy-chain V region occurs in two separate stages. In the first, a D_H gene segment is joined to a J_H gene segment; then a V_H gene segment rearranges to DJ_H to make a complete V_H-region exon. As with the light-chain genes, RNA splicing joins the assembled V-region sequence to the neighboring C-region gene.
• Diversification of the antibody repertoire occurs in two stages: primarily by rearrangement (“V(D)J recombination”) of Ig V, D and J gene segments in precursor B cells resident in the bone marrow, and then by somatic mutation and class switch recombination of these rearranged Ig genes when mature B cells are activated. Immunoglobulin somatic mutation and class switching are central to the maturation of the immune response and the generation of a “memory” response.
• The genomic loci of antibodies are very large and they are located on different chromosomes. The immunoglobulin gene segments are organized into three clusters or genetic loci: the κ, λ, and heavy-chain loci. Each is organized slightly differently. For example, in humans, immunoglobulin genes are organized as follows. The λ light-chain locus is located on chromosome 22 and a cluster of V_λ gene segments is followed by four sets of J_k gene segments each linked to a single C_λ gene. The κ light-chain locus is on chromosome 2 and the cluster of V_κ gene segments is followed by a cluster of J_κ gene segments, and then by a single C_κ gene. The organization of the heavy-chain locus, on chromosome 14, resembles that of the κ locus, with separate clusters of V_H, D_H, and J_H gene segments and of C_H genes. The heavy-chain locus differs in one important way: instead of a single C-region, it contains a series of C regions arrayed one after the other, each of which corresponds to a different isotype. There are five immunoglobulin heavy chain isotypes: IgM, IgG, IgA, IgE and IgD. Generally, a cell expresses only one at a time, beginning with IgM. The expression of other isotypes, such as IgG, can occur through isotype switching.
• The joining of various V, D and J genes is an entirely random event that results in approximately 50,000 different possible combinations for VDJ(H) and approximately 1,000 for VJ(L). Subsequent random pairing of H and L chains brings the total number of antibody specificities to about 10⁷ possibilities. Diversity is further increased by the imprecise joining of different genetic segments. Rearrangements occur on both DNA strands, but only one strand is transcribed (due to allelic exclusion). Only one rearrangement occurs in the life of a B cell because of irreversible deletions in DNA. Consequently, each mature B cell maintains one immunologic specificity and is maintained in the progeny or clone. This constitutes the molecular basis of the clonal selection; i.e., each antigenic determinant triggers the response of the pre-existing clone of B lymphocytes bearing the specific receptor molecule. The primary repertoire of B cells, which is established by V(D)J recombination, is primarily controlled by two closely linked genes, recombination activating gene (RAG)-1 and RAG-2.
• Over the last decade, considerable diversity among vertebrates in both Ig gene diversity and antibody repertoire development has been revealed. Rodents and humans have five heavy chain classes, IgM, IgD, IgG, IgE and IgA, and each have four subclasses of IgG and one or two subclasses of IgA, while rabbits have a single IgG heavy chain gene but 13 genes for different IgA subclasses (Burnett, R. O et al. EMBO J 8:4047; Honjo, In Honjo, T, Alt. F. W. T. H. eds, Immunoglobulin Genes p. 123 Academic Press, New York). Swine have at least six IgG subclasses (Kacskovics, I et al. 1994 J Immunol 153:3565), but no IgD (Butler et al. 1996 Inter. Immunol 8:1897-1904). A gene encoding IgD has only been described in rodents and primates. Diversity in the mechanism of repertoire development is exemplified by contrasting the pattern seen in rodents and primates with that reported for chickens, rabbits, swine and the domesticated Bovidae. Whereas the former group have a large number of V_H genes belonging to seven to 10 families (Rathbun, G. In Hongo, T. Alt. F. W. and Rabbitts, T. H., eds, Immunoglobulin Genes, p. 63, Academic press New York), the V_H genes of each member of the latter group belong to a single V_H gene family (Sun, J. et al. 1994 J. Immunol. 1553:56118; Dufour, V et al. 1996, J Immunol. 156:2163). With the exception of the rabbit, this family is composed of less than 25 genes. Whereas rodents and primates can utilize four to six J_H segments, only a single J_H is available for repertoire development in the chicken (Reynaud et al. 1989 Adv. Immunol. 57:353). Similarly, Butler et al. (1996 Inter. Immunol 8:1897-1904) hypothesized that swine may resemble the chicken in having only a single J_H gene. These species generally have fewer V, D and J genes; in the pig and cow a single VH gene family exists, consisting of less than 20 gene segments (Butler et al, Advances in Swine in Biomedical Research, eds: Tumbleson and Schook, 1996; Sinclair et al, J. Immunol. 159: 3883, 1997). Together with lower numbers of J and D gene segments, this results in significantly less diversity being generated by gene rearrangement. However, there does appear to be greater numbers of light chain genes in these species. Similar to humans and mice, these species express a single κ light chain but multiple λ light chain genes. However, these do not seem to affect the restricted diversity that is achieved by rearrangement.
• Since combinatorial joining of more than 100 V_H, 20-30 D_H and four to six J_H gene segments is a major mechanism of generating the antibody repertoire in humans, species with fewer V_H, D_H or J_H segments must either generate a smaller repertoire or use alternative mechanisms for repertoire development. Ruminants, pigs, rabbits and chickens, utilize several mechanisms to generate antibody diversity. In these species there appears to be an important secondary repertoire development, which occurs in highly specialized lymphoid tissue such as ileal Peyer's patches (Binns and Licence, Adv. Exp. Med. Biol. 186: 661, 1985). Secondary repertoire development occurs in these species by a process of somatic mutation which is a random and not fully understood process. The mechanism for this repertoire diversification appears to be templated mutation, or gene conversion (Sun et al, J. Immunol. 153: 5618, 1994) and somatic hypermutation.
• Gene conversion is important for antibody diversification in some higher vertebrates, such as chickens, rabbits and cows. In mice, however, conversion events appear to be infrequent among endogenous antibody genes. Gene conversion is a distinct diversifying mechanism characterized by transfers of homologous sequences from a donor antibody V gene segment to an acceptor V gene segment. If donor and acceptor segments have numerous sequence differences then gene conversion can introduce a set of sequence changes into a V region by a single event. Depending on the species, gene conversion events can occur before and/or after antigen exposure during B cell differentiation (Tsai et al. International Immunology, Vol. 14, No. 1, 55-64, January 2002).
• Somatic hypermutation achieves diversification of antibody genes in all higher vertebrate species. It is typified by the introduction of single point mutations into antibody V(D)J segments. Generally, hypermutation appears to be activated in B cells by antigenic stimulation.
• Production of Animals with Humanized Immune Systems
• In order to reduce the immunogenicity of antibodies generated in mice for human therapeutics, various attempts have been made to replace murine protein sequences with human protein sequences in a process now known as humanization. Transgenic mice have been constructed which have had their own immunoglobulin genes functionally replaced with human immunoglobulin genes so that they produce human antibodies upon immunization. Elimination of mouse antibody production was achieved by inactivation of mouse Ig genes in embryonic stem (ES) cells by using gene-targeting technology to delete crucial cis-acting sequences involved in the process of mouse Ig gene rearrangement and expression. B cell development in these mutant mice could be restored by the introduction of megabase-sized YACs containing a human germline-configuration H- and κ L-chain minilocus transgene. The expression of fully human antibody in these transgenic mice was predominant, at a level of several 100 μg/l of blood. This level of expression is several hundred-fold higher than that detected in wild-type mice expressing the human Ig gene, indicating the importance of inactivating the endogenous mouse Ig genes in order to enhance human antibody production by mice.
• The first humanization attempts utilized molecular biology techniques to construct recombinant antibodies. For example, the complementarity determining regions (CDR) from a mouse antibody specific for a hapten were grafted onto a human antibody framework, effecting a CDR replacement. The new antibody retained the binding specificity conveyed by the CDR sequences (P. T. Jones et al. Nature 321: 522-525 (1986)). The next level of humanization involved combining an entire mouse VH region with a human constant region such as gamma₁ (S. L. Morrison et al., Proc. Natl. Acad. Sci., 81, pp. 6851-6855 (1984)). However, these chimeric antibodies, which still contain greater than 30% xenogeneic sequences, are sometimes only marginally less immunogenic than totally xenogeneic antibodies (M. Bruggemann et al., J. Exp. Med., 170, pp. 2153-2157 (1989)).
• Subsequently, attempts were carried out to introduce human immunoglobulin genes into the mouse, thus creating transgenic mice capable of responding to antigens with antibodies having human sequences (Bruggemann et al. Proc. Nat'l. Acad. Sci. USA 86:6709-6713 (1989)). Due to the large size of human immunoglobulin genomic loci, these attempts were thought to be limited by the amount of DNA, which could be stably maintained by available cloning vehicles. As a result, many investigators concentrated on producing mini-loci containing limited numbers of V region genes and having altered spatial distances between genes as compared to the natural or germline configuration (See, for example, U.S. Pat. No. 5,569,825). These studies indicated that producing human sequence antibodies in mice was possible, but serious obstacles remained regarding obtaining sufficient diversity of binding specificities and effector functions (isotypes) from these transgenic animals to meet the growing demand for antibody therapeutics.
• In order to provide additional diversity, work has been conducted to add large germline fragments of the human Ig locus into transgenic mammals. For example, a majority of the human V, D, and J region genes arranged with the same spacing found in the unrearranged germline of the human genome and the human Cμ and Cδ constant regions was introduced into mice using yeast artificial chromosome (YAC) cloning vectors (See, for example, WO 94/02602). A 22 kb DNA fragment comprising sequences encoding a human gamma-2 constant region and the upstream sequences required for class-switch recombination was latter appended to the foregoing transgene. In addition, a portion of a human kappa locus comprising Vκ, Jκ and Cκ region genes, also arranged with substantially the same spacing found in the unrearranged germline of the human genome, was introduced into mice using YACS. Gene targeting was used to inactivate the murine IgH & kappa light chain immunoglobulin gene loci and such knockout strains were bred with the above transgenic strains to generate a line of mice having the human V, D, J, Cμ, Cδ, and Cγ₂ constant regions as well as the human Vκ, Jκ and Cκ region genes all on an inactivated murine immunoglobulin background (See, for example, PCT patent application WO 94/02602 to Kucherlapati et al.; see also Mendez et al., Nature Genetics 15:146-156 (1997)).
• Yeast artificial chromosomes as cloning vectors in combination with gene targeting of endogenous loci and breeding of transgenic mouse strains provided one solution to the problem of antibody diversity. Several advantages were obtained by this approach. One advantage was that YACs can be used to transfer hundreds of kilobases of DNA into a host cell. Therefore, use of YAC cloning vehicles allows inclusion of substantial portions of the entire human Ig heavy and light chain regions into a transgenic mouse thus approaching the level of potential diversity available in the human. Another advantage of this approach is that the large number of V genes has been shown to restore full B cell development in mice deficient in murine immunoglobulin production. This ensures that these reconstituted mice are provided with the requisite cells for mounting a robust human antibody response to any given immunogen. (See, for example, WO 94/02602; L. Green and A. Jakobovits, J. Exp. Med. 188:483-495 (1998)). A further advantage is that sequences can be deleted or inserted onto the YAC by utilizing high frequency homologous recombination in yeast. This provides for facile engineering of the YAC transgenes.
• In addition, Green et al. Nature Genetics 7:13-21 (1994) describe the generation of YACs containing 245 kb and 190 kb-sized germline configuration fragments of the human heavy chain locus and kappa light chain locus, respectively, which contained core variable and constant region sequences. The work of Green et al. was recently extended to the introduction of greater than approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and kappa light chain loci, respectively, to produce XenoMouse™ mice. See, for example, Mendez et al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), European Patent No. EP 0 463 151 B1, PCT Publication Nos. WO 94/02602, WO 96/34096 and WO 98/24893.
• Several strategies exist for the generation of mammals that produce human antibodies. In particular, there is the “minilocus” approach that is typified by work of GenPharm International, Inc. and the Medical Research Council, YAC introduction of large and substantially germline fragments of the Ig loci that is typified by work of Abgenix, Inc. (formerly Cell Genesys). The introduction of entire or substantially entire loci through the use microcell fusion as typified by work of Kirin Beer Kabushiki Kaisha.
• In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V_H genes, one or more D_H genes, one or more J_H genes, a mu constant region, and a second constant region (such as a gamma constant region) are formed into a construct for insertion into an animal. See, for example, U.S. Pat. Nos. 5,545,807, 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, 5,643,763; European Patent No. 0 546 073; PCT Publication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884; Taylor et al. Nucleic Acids Research 20:6287-6295 (1992), Chen et al. International Immunology 5:647-656 (1993), Tuaillon et al. J. Immunol. 154:6453-6465 (1995), Choi et al. Nature Genetics 4:117-123 (1993), Lonberg et al. Nature 368:856-859 (1994), Taylor et al. International Immunology 6:579-591 (1994), Tuaillon et al. J. Immunol. 154:6453-6465 (1995), and Fishwild et al. Nature Biotech. 14:845-851 (1996).
• In the microcell fusion approach, portions or whole human chromosomes can be introduced into mice (see, for example, European Patent Application No. EP 0 843 961 A1). Mice generated using this approach and containing the human Ig heavy chain locus will generally possess more than one, and potentially all, of the human constant region genes. Such mice will produce, therefore, antibodies that bind to particular antigens having a number of different constant regions.
• While mice remain the most developed animal for the expression of human immunoglobulins in humans, recent technological advances have allowed for progress to begin in applying these techniques to other animals, such as cows. The general approach in mice has been to genetically modify embryonic stem cells of mice to knock-out murine immunoglobulins and then insert YACs containing human immunoglobulins into the ES cells. However, ES cells are not available for cows or other large animals such as sheep and pigs. Thus, several fundamental developments had to occur before even the possibility existed to generate large animals with immunoglobulin genes knocked-out and that express human antibody. The alternative to ES cell manipulation to create genetically modified animals is cloning using somatic cells that have been genetically modified. Cloning using genetically modified somatic cells for nuclear transfer has only recently been accomplished.
• Since the announcement of Dolly's (a cloned sheep) birth from an adult somatic cell in 1997 (Wilmut, I., et al (1997) Nature 385: 810-813), ungulates, including cattle (Cibelli, J et al 1998 Science 280: 1266-1258; Kubota, C. et al. 2000 Proc. Nat'l. Acad. Sci 97: 990-995), goats (Baguisi, A. et al., (1999) Nat. Biotechnology 17: 456-461), and pigs (Polejaeva, I. A., et al. 2000 Nature 407: 86-90; Betthauser, J. et al. 2000 Nat. Biotechnology 18: 1055-1059) have been cloned.
• The next technological advance was the development of the technique to genetically modify the cells prior to nuclear transfer to produce genetically modified animals. PCT publication No. WO 00/51424 to PPL Therapeutics describes the targeted genetic modification of somatic cells for nuclear transfer.
• Subsequent to these fundamental developments, single and double allele knockouts of genes and the birth of live animals with these modifications have been reported. Between 2002 and 2004, three independent groups, Immerge Biotherapeutics, Inc. in collaboration with the University of Missouri (Lai et al. (Science (2002) 295: 1089-1092) & Kolber-Simonds et al. (PNAS. (2004) 101(19):7335-40)), Alexion Pharmaceuticals (Ramsoondar et al. (Biol Reprod (2003)69: 437-445) and Revivicor, Inc. (Dai et al. (Nature Biotechnology (2002) 20: 251-255) & Phelps et al. (Science (2003) January 17; 299(5605):411-4)) produced pigs that lacked one allele or both alleles of the alpha-1,3-GT gene via nuclear transfer from somatic cells with targeted genetic deletions. In 2003, Sedai et al. (Transplantation (2003) 76:900-902) reported the targeted disruption of one allele of the alpha-1,3-GT gene in cattle, followed by the successful nuclear transfer of the nucleus of the genetically modified cell and production of transgenic fetuses.
• Thus, the feasibility of knocking-out immunoglobulin genes in large animals and inserting human immunoglobulin loci into their cells is just now beginning to be explored. However, due to the complexity and species differences of immunoglobulin genes, the genomic sequences and arrangement of Ig kappa, lambda and heavy chains remain poorly understood in most species. For example, in pigs, partial genomic sequence and organization has only been described for heavy chain constant alpha, heavy chain constant mu and heavy chain constant delta (Brown and Butler Mol Immunol. 1994 June; 31(8):633-42, Butler et al Vet Immunol Immunopathol. 1994 October; 43(1-3):5-12, and Zhao et al J Immunol. 2003 Aug. 1; 171(3):1312-8).
• In cows, the immunoglobulin heavy chain locus has been mapped (Zhao et al. 2003 J. Biol. Chem. 278:35024-32) and the cDNA sequence for the bovine kappa gene is known (See, for example, U.S. Patent Publication No. 2003/0037347). Further, approximately 4.6 kb of the bovine mu heavy chain locus has been sequenced and transgenic calves with decreased expression of heavy chain immunoglobulins have been created by disrupting one or both alleles of the bovine mu heavy chain. In addition, a mammalian artificial chromosome (MAC) vector containing the entire unarranged sequences of the human Ig H-chain and κ L-chain has been introduced into cows (TC cows) with the technology of microcell-mediated chromosome transfer and nuclear transfer of bovine fetal fibroblast cells (see, for example, Kuroiwa et al. 2002 Nature Biotechnology 20:889, Kuroiwa et al. 2004 Nat Genet. June 6 Epub, U.S. Patent Publication Nos. 2003/0037347, 2003/0056237, 2004/0068760 and PCT Publication No. WO 02/07648).
• While significant progress has been made in the production of bovine that express human immunoglobulin, little has been accomplished in other large animals, such as sheep, goats and pigs. Although cDNA sequence information for immunoglobulin genes of sheep, goats and pigs is readily available in Genbank, the unique nature of immunoglobulin loci, which undergo massive rearrangements, creates the need to characterize beyond sequences known to be present in mRNAs (or cDNAs). Since immunoglobulin loci are modular and the coding regions are redundant, deletion of a known coding region does not ensure altered function of the locus. For example, if one were to delete the coding region of a heavy-chain variable region, the function of the locus would not be significantly altered because hundreds of other function variable genes remain in the locus. Therefore, one must first characterize the locus to identify a potential “Achilles heel”.
• Despite some advancements in expressing human antibodies in cattle, greater challenges remain for inactivation of the endogenous bovine Ig genes, increasing expression levels of the human antibodies and creating human antibody expression in other large animals, such as porcine, for which the sequence and arrangement of immunoglobulin genes are largely unknown.
• It is therefore an object of the present invention to provide the arrangement of ungulate immunoglobin germline gene sequence.
• It is another object of the present invention to provide novel ungulate immunoglobulin genomic sequences.
• It is a further object of the present invention to provide cells, tissues and animals lacking at least one allele of a heavy and/or light chain immunoglobulin gene.
• It is another object of the present invention to provide ungulates that express human immunoglobulins.
• It is a still further object of the present invention to provide methods to generate cells, tissues and animals lacking at least one allele of novel ungulate immunoglobulin gene sequences and/or express human immunoglobulins.
SUMMARY OF THE INVENTION
• The present invention provides for the first time ungulate immunoglobin germline gene sequence arrangement as well as novel genomic sequences thereof. In addition, novel ungulate cells, tissues and animals that lack at least one allele of a heavy or light chain immunoglobulin gene are provided. Based on this discovery, ungulates can be produced that completely lack at least one allele of a heavy and/or light chain immunoglobulin gene. In addition, these ungulates can be further modified to express xenogenous, such as human, immunoglobulin loci or fragments thereof.
• In one aspect of the present invention, a transgenic ungulate that lacks any expression of functional endogenous immunoglobulins is provided. In one embodiment, the ungulate can lack any expression of endogenous heavy and/or light chain immunoglobulins. The light chain immunoglobulin can be a kappa and/or lambda immunoglobulin. In additional embodiments, transgenic ungulates are provided that lack expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof. In one embodiment, the expression of functional endogenous immunoglobulins can be accomplished by genetic targeting of the endogenous immunoglobulin loci to prevent expression of the endogenous immunoglobulin. In one embodiment, the genetic targeting can be accomplished via homologous recombination. In another embodiment, the transgenic ungulate can be produced via nuclear transfer.
• In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogenous immunoglobulin locus. In one embodiment, the xenogenous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
• In another aspect of the present invention, transgenic ungulates are provided that expresses a xenogenous immunoglobulin loci or fragment thereof, wherein the immunoglobulin can be expressed from an immunoglobulin locus that is integrated within an endogenous ungulate chromosome. In one embodiment, ungulate cells derived from the transgenic animals are provided. In one embodiment, the xenogenous immunoglobulin locus can be inherited by offspring. In another embodiment, the xenogenous immunoglobulin locus can be inherited through the male germ line by offspring. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
• In another aspect of the present invention, novel genomic sequences encoding the heavy chain locus of ungulate immunoglobulin are provided. In one embodiment, an isolated nucleotide sequence encoding porcine heavy chain is provided that includes at least one variable region, two diversity regions, at least four joining regions and at least one constant region, such as the mu constant region, for example, as represented in Seq ID No. 29. In another embodiment, an isolated nucleotide sequence is provided that includes at least four joining regions and at least one constant region, such as the mu constant region, of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No. 4. In a further embodiment, nucleotide sequence is provided that includes 5′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No 1. Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in the 3′ region of Seq ID No 4. In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 1, 4 or 29 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 1, 4 or 29 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. In one embodiment, the nucleotide sequence contains at least 17, 20, 25 or 30 contiguous nucleotides of Seq ID No 4 or residues 1-9,070 of Seq ID No 29. In another embodiment, the nucleotide sequence contains residues 9,070-11039 of Seq ID No 29. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1, 4 or 29, as well as, nucleotides homologous thereto.
• In another embodiment, novel genomic sequences encoding the kappa light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate kappa light chain regions. In one embodiment, nucleic acid sequence is provided that encodes the porcine kappa light chain locus. In another embodiment, the nucleic acid sequence can contain at least one joining region, one constant region and/or one enhancer region of kappa light chain. In a further embodiment, the nucleotide sequence can include at least five joining regions, one constant region and one enhancer region, for example, as represented in Seq ID No. 30. In a further embodiment, an isolated nucleotide sequence is provided that contains at least one, at least two, at least three, at least four or five joining regions and 3′ flanking sequence to the joining region of porcine genomic kappa light chain, for example, as represented in Seq ID No 12. In another embodiment, an isolated nucleotide sequence of porcine genomic kappa light chain is provided that contains 5′ flanking sequence to the first joining region, for example, as represented in Seq ID No 25. In a further embodiment, an isolated nucleotide sequence is provided that contains 3′ flanking sequence to the constant region and, optionally, the 5′ portion of the enhancer region, of porcine genomic kappa light chain, for example, as represented in Seq ID Nos. 15, 16 and/or 19.
• In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 30, 12, 25, 15, 16 or 19 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 30, 12, 25, 15, 16 or 19, as well as, nucleotides homologous thereto.
• In another embodiment, novel genomic sequences encoding the lambda light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate lambda light chain regions. In one embodiment, the porcine lambda light chain nucleotides include a concatamer of J to C units. In a specific embodiment, an isolated porcine lambda nucleotide sequence is provided, such as that depicted in Seq ID No. 28. In one embodiment, a nucleotide sequence is provided that includes 5′ flanking sequence to the first lambda J/C unit of the porcine lambda light chain genomic sequence, for example, as represented by Seq ID No 32. Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, approximately 200 base pairs downstream of lambda J/C, such as that represented by Seq ID No 33. Alternatively, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, as represented by Seq ID No 34, 35, 36, 37, 38, and/or 39.
• In a further embodiment, nucleic acid sequences are provided that encode bovine lambda light chain locus, which can include at least one joining region-constant region pair and/or at least one variable region, for example, as represented by Seq ID No. 31. In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39, as well as, nucleotides homologous thereto.
• In another embodiment, nucleic acid targeting vector constructs are also provided. The targeting vectors can be designed to accomplish homologous recombination in cells. These targeting vectors can be transformed into mammalian cells to target the ungulate heavy chain, kappa light chain or lambda light chain genes via homologous recombination. In one embodiment, the targeting vectors can contain a 3′ recombination arm and a 5′ recombination arm (i.e. flanking sequence) that is homologous to the genomic sequence of ungulate heavy chain, kappa light chain or lambda light chain genomic sequence, for example, sequence represented by Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The homologous DNA sequence can include at least 15 bp, 20 bp, 25 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence homologous to the genomic sequence.
• In one embodiment, the 5′ and 3′ recombination arms of the targeting vector can be designed such that they flank the 5′ and 3′ ends of at least one functional variable, joining, diversity, and/or constant region of the genomic sequence. The targeting of a functional region can render it inactive, which results in the inability of the cell to produce functional immunoglobulin molecules. In another embodiment, the homologous DNA sequence can include one or more intron and/or exon sequences. In addition to the nucleic acid sequences, the expression vector can contain selectable marker sequences, such as, for example, enhanced Green Fluorescent Protein (eGFP) gene sequences, initiation and/or enhancer sequences, poly A-tail sequences, and/or nucleic acid sequences that provide for the expression of the construct in prokaryotic and/or eukaryotic host cells. The selectable marker can be located between the 5′ and 3′ recombination arm sequence.
• In one particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the J6 region of the porcine immunoglobulin heavy chain locus. Since the J6 region is the only functional joining region of the porcine immunoglobulin heavy chain locus, this will prevent the expression of a functional porcine heavy chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the J6 region, including J1-4, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the J6 region, including the mu constant region (a “J6 targeting construct”), see for example, FIG. 1. Further, this J6 targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No 5 and FIG. 1. In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the diversity region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the diversity region of the porcine heavy chain locus. In a further embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the mu constant region and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the mu constant region of the porcine heavy chain locus.
• In another particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the constant region of the porcine immunoglobulin kappa light chain locus. Since the present invention discovered that there is only one constant region of the porcine immunoglobulin kappa light chain locus, this will prevent the expression of a functional porcine kappa light chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the constant region, optionally including the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the constant region, optionally including at least part of the enhancer region (a “Kappa constant targeting construct”), see for example, FIG. 2. Further, this kappa constant targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No 20 and FIG. 2. In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the joining region of the porcine kappa light chain locus.
• In another particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the J/C region of the porcine lambda light chain. See FIG. 3. Disruption of the J/C region will prevent the expression of a functional porcine kappa light chain immunoglobulin. In one embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the first J/C unit and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the last J/C unit. Further, this lambda light chain targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example FIG. 4.
• In a further embodiment, more than one targeting vector can be used to target the ungulate heavy chain, kappa light chain or lambda light chain genes via homologous recombination. For example, two targeting vectors can be used to target the gene of interest. A first targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 5′ flanking sequence of at least one functional variable, joining, diversity, and/or constant region of the genomic sequence. A second targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ flanking sequence at least one functional variable, joining, diversity, and/or constant region of the genomic sequence.
• In a particular embodiment, the first targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 5′ flanking sequence of the first J/C unit in the J/C cluster region. See FIG. 5. According to this embodiment, a second targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ flanking sequence of the last J/C unit in the J/C cluster region. See FIG. 6.
• In another embodiment, primers are provided to generate 3′ and 5′ sequences of a targeting vector. The oligonucleotide primers can be capable of hybridizing to porcine immunoglobulin genomic sequence, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. In a particular embodiment, the primers hybridize under stringent conditions to Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. Another embodiment provides oligonucleotide probes capable of hybridizing to porcine heavy chain, kappa light chain or lambda light chain nucleic acid sequences, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The polynucleotide primers or probes can have at least 14 bases, 20 bases, 30 bases, or 50 bases which hybridize to a polynucleotide of the present invention. The probe or primer can be at least 14 nucleotides in length, and in a particular embodiment, are at least 15, 20, 25, 28, or 30 nucleotides in length.
• In one embodiment, primers are provided to amplify a fragment of porcine Ig heavy-chain that includes the functional joining region (the J6 region). In one non-limiting embodiment, the amplified fragment of heavy chain can be represented by Seq ID No 4 and the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 2, to produce the 5′ recombination arm and complementary to a portion of Ig heavy-chain mu constant region, such as, but not limited to Seq ID No 3, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 4) can be subcloned and assembled into a targeting vector.
• In other embodiments, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the constant region. In another embodiment, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the J region. In one non-limiting embodiment, the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 21 or 10, to produce the 5′ recombination arm and complementary to genomic sequence 3′ of the constant region, such as, but not limited to Seq ID No 14, 24 or 18, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 20) can be subcloned and assembled into a targeting vector.
• In another aspect of the present invention, ungulate cells lacking at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the process, sequences and/or constructs described herein are provided. These cells can be obtained as a result of homologous recombination. Particularly, by inactivating at least one allele of an ungulate heavy chain, kappa light chain or lambda light chain gene, cells can be produced which have reduced capability for expression of ungulate antibodies. In other embodiments, mammalian cells lacking both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be produced according to the process, sequences and/or constructs described herein. In a further embodiment, porcine animals are provided in which at least one allele of an ungulate heavy chain, kappa light chain and/or lambda light chain gene is inactivated via a genetic targeting event produced according to the process, sequences and/or constructs described herein. In another aspect of the present invention, porcine animals are provided in which both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene are inactivated via a genetic targeting event. The gene can be targeted via homologous recombination.
• In other embodiments, the gene can be disrupted, i.e. a portion of the genetic code can be altered, thereby affecting transcription and/or translation of that segment of the gene. For example, disruption of a gene can occur through substitution, deletion (“knock-out”) or insertion (“knock-in”) techniques. Additional genes for a desired protein or regulatory sequence that modulate transcription of an existing sequence can be inserted. To achieve multiple genetic modifications of ungulate immunoglobulin genes, in one embodiment, cells can be modified sequentially to contain multiple genetic modifications. In other embodiments, animals can be bred together to produce animals that contain multiple genetic modifications of immunoglobulin genes. As an illustrative example, animals that lack expression of at least one allele of an ungulate heavy chain gene can be further genetically modified or bred with animals lacking at least one allele of a kappa light chain gene.
• In embodiments of the present invention, alleles of ungulate heavy chain, kappa light chain or lambda light chain gene are rendered inactive according to the process, sequences and/or constructs described herein, such that functional ungulate immunoglobulins can no longer be produced. In one embodiment, the targeted immunoglobulin gene can be transcribed into RNA, but not translated into protein. In another embodiment, the targeted immunoglobulin gene can be transcribed in an inactive truncated form. Such a truncated RNA may either not be translated or can be translated into a nonfunctional protein. In an alternative embodiment, the targeted immunoglobulin gene can be inactivated in such a way that no transcription of the gene occurs. In a further embodiment, the targeted immunoglobulin gene can be transcribed and then translated into a nonfunctional protein.
• In a further aspect of the present invention, ungulate, such as porcine or bovine, cells lacking one allele, optionally both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be used as donor cells for nuclear transfer into recipient cells to produce cloned, transgenic animals. Alternatively, ungulate heavy chain, kappa light chain and/or lambda light chain gene knockouts can be created in embryonic stem cells, which are then used to produce offspring. Offspring lacking a single allele of a functional ungulate heavy chain, kappa light chain and/or lambda light chain gene produced according to the process, sequences and/or constructs described herein can be breed to further produce offspring lacking functionality in both alleles through mendelian type inheritance.
• In one aspect of the present invention, a method is provided to disrupt the expression of an ungulate immunoglobulin gene by (i) analyzing the germline configuration of the ungulate heavy chain, kappa light chain or lambda light chain genomic locus; (ii) determining the location of nucleotide sequences that flank the 5′ end and the 3′ end of at least one functional region of the locus; and (iii) transfecting a targeting construct containing the flanking sequence into a cell wherein, upon successful homologous recombination, at least one functional region of the immunoglobulin locus is disrupted thereby reducing or preventing the expression of the immunoglobulin gene. In one embodiment, the germline configuration of the porcine heavy chain locus is provided. The porcine heavy chain locus contains at least four variable regions, two diversity regions, six joining regions and five constant regions, for example, as illustrated in FIG. 1. In a specific embodiment, only one of the six joining regions, J6, is functional. In another embodiment, the germline configuration of the porcine kappa light chain locus is provided. The porcine kappa light chain locus contains at least six variable regions, six joining regions, one constant region and one enhancer region, for example, as illustrated in FIG. 2. In a further embodiment, the germline configuration of the porcine lambda light chain locus is provided. The porcine lambda light chain locus contains a variable region and the J/C region. See FIG. 3.
• In a further aspect of the present invention, a method is provided to disrupt the expression of an ungulate lambda light chain locus by (i) analyzing the germline configuration of the ungulate lambda light chain genomic locus; (ii) determining the location of nucleotide sequences that flank the 5′ end of at least one functional region of the locus; (ii) constructing a 5′ targeting construct; (iv) determining the location of nucleotide sequences that flank the 3′ end of at least one functional region of the locus; (v) constructing a 3′ targeting construct; (vi) transfecting both the 5′ and the 3′ targeting constructs into a cell wherein, upon successful homologous recombination of each targeting construct, at least one functional region of the immunoglobulin locus is disrupted thereby reducing or preventing the expression of the immunoglobulin gene. See FIGS. 5 and 6.
• In one embodiment, the germline configuration of the porcine lambda light chain locus is provided. The porcine lambda light chain locus contains a variable region and a J/C region. See FIG. 3.
• In further aspects of the present invention provides ungulates and ungulate cells that lack at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the processes, sequences and/or constructs described herein, which are further modified to express at least part of a human antibody (i.e. immunoglobulin (Ig)) locus. In additional embodiments, porcine animals are provided that express xenogenous immunoglobulin. This human locus can undergo rearrangement and express a diverse population of human antibody molecules in the ungulate. These cloned, transgenic ungulates provide a replenishable, theoretically infinite supply of human antibodies (such as polyclonal antibodies), which can be used for therapeutic, diagnostic, purification, and other clinically relevant purposes. In one particular embodiment, artificial chromosomes (ACs), such as yeast or mammalian artificial chromosomes (YACS or MACS) can be used to allow expression of human immunoglobulin genes into ungulate cells and animals. All or part of human immunoglobulin genes, such as the Ig heavy chain gene (human chromosome 414), Ig kappa chain gene (human chromosome #2) and/or the Ig lambda chain gene (chromosome #22) can be inserted into the artificial chromosomes, which can then be inserted into ungulate cells. In further embodiments, ungulates and ungulate cells are provided that contain either part or all of at least one human antibody gene locus, which undergoes rearrangement and expresses a diverse population of human antibody molecules.
• In additional embodiments, methods of producing xenogenous antibodies are provided, wherein the method can include: (a) administering one or more antigens of interest to an ungulate whose cells comprise one or more artificial chromosomes and lack any expression of functional endogenous immunoglobulin, each artificial chromosome comprising one or more xenogenous immunoglobulin loci that undergo rearrangement, resulting in production of xenogenous antibodies against the one or more antigens; and/or (b) recovering the xenogenous antibodies from the ungulate. In one embodiment, the immunoglobulin loci can undergo rearrangement in a B cell.
• In one aspect of the present invention, an ungulate, such as a pig or a cow, can be prepared by a method in accordance with any aspect of the present invention. These cloned, transgenic ungulates (e.g., porcine and bovine animals) provide a replenishable, theoretically infinite supply of human polyclonal antibodies, which can be used as therapeutics, diagnostics and for purification purposes. For example, transgenic animals produced according to the process, sequences and/or constructs described herein that produce polyclonal human antibodies in the bloodstream can be used to produce an array of different antibodies which are specific to a desired antigen. The availability of large quantities of polyclonal antibodies can also be used for treatment and prophylaxis of infectious disease, vaccination against biological warfare agents, modulation of the immune system, removal of undesired human cells such as cancer cells, and modulation of specific human molecules.
• In other embodiments, animals or cells lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can contain additional genetic modifications to eliminate the expression of xenoantigens. Such animals can be modified to eliminate the expression of at least one allele of the alpha-1,3-galactosyltransferase gene, the CMP-Neu5Ac hydroxylase gene (see, for example, U.S. Ser. No. 10/863,116), the iGb3 synthase gene (see, for example, U.S. Patent Application 60/517,524), and/or the Forssman synthase gene (see, for example, U.S. Patent Application 60/568,922). In additional embodiments, the animals discloses herein can also contain genetic modifications to express fucosyltransferase and/or sialyltransferase. To achieve these additional genetic modifications, in one embodiment, cells can be modified to contain multiple genetic modifications. In other embodiments, animals can be bred together to achieve multiple genetic modifications. In one specific embodiment, animals, such as pigs, lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can be bred with animals, such as pigs, lacking expression of alpha-1,3-galactosyl transferase (for example, as described in WO 04/028243).
BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1 illustrates the design of a targeting vector that disrupts the expression of the joining region of the porcine heavy chain immunoglobulin gene.
• FIG. 2 illustrates the design of a targeting vector that disrupts the expression of the constant region of the porcine kappa light chain immunoglobulin gene.
• FIG. 3 illustrates the genomic organization of the porcine lambda immunoglobulin locus, including a concatamer of J-C sequences or units as well as flanking regions that include the variable region 5′ to the JC cluster region. Bacterial artificial chromosomes (BAC1 and BAC2) represent fragments of the porcine immunoglobulin genome that can be obtained from BAC libraries.
• FIG. 4 represents the design of a targeting vector that disrupts the expression of the JC cluster region of the porcine lambda light chain immunoglobulin gene. “SM” stands for a selectable marker gene, which can be used in the targeting vector.
• FIG. 5 illustrates a targeting strategy to insert a site specific recombinase target or recognition site into the region 5′ of the JC cluster region of the porcine lambda immunoglobulin locus. “SM” stands for a selectable marker gene, which can be used in the targeting vector. “SSRRS” stands for a specific recombinase target or recognition site.
• FIG. 6 illustrates a targeting strategy to insert a site specific recombinase target or recognition site into the region 3′ of the JC cluster region of the porcine lambda immunoglobulin locus. “SM” stands for a selectable marker gene, which can be used in the targeting vector. “SSRRS” stands for a specific recombinase target or recognition site.
• FIG. 7 illustrates the site specific recombinase mediated transfer of a YAC into a host genome. “SSRRS” stands for a specific recombinase target or recognition site.
DETAILED DESCRIPTION
• The present invention provides for the first time ungulate immunoglobin germline gene sequence arrangement as well as novel genomic sequences thereof. In addition, novel ungulate cells, tissues and animals that lack at least one allele of a heavy or light chain immunoglobulin gene are provided. Based on this discovery, ungulates can be produced that completely lack at least one allele of a heavy and/or light chain immunoglobulin gene. In addition, these ungulates can be further modified to express xenogenous, such as human, immunoglobulin loci or fragments thereof.
• In one aspect of the present invention, a transgenic ungulate that lacks any expression of functional endogenous immunoglobulins is provided. In one embodiment, the ungulate can lack any expression of endogenous heavy and/or light chain immunoglobulins. The light chain immunoglobulin can be a kappa and/or lambda immunoglobulin. In additional embodiments, transgenic ungulates are provided that lack expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof. In one embodiment, the expression of functional endogenous immunoglobulins can be accomplished by genetic targeting of the endogenous immunoglobulin loci to prevent expression of the endogenous immunoglobulin. In one embodiment, the genetic targeting can be accomplished via homologous recombination. In another embodiment, the transgenic ungulate can be produced via nuclear transfer.
• In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogenous immunoglobulin locus. In one embodiment, the xenogenous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
• In another aspect of the present invention, transgenic ungulates are provided that expresses a xenogenous immunoglobulin loci or fragment thereof, wherein the immunoglobulin can be expressed from an immunoglobulin locus that is integrated within an endogenous ungulate chromosome. In one embodiment, ungulate cells derived from the transgenic animals are provided. In one embodiment, the xenogenous immunoglobulin locus can be inherited by offspring. In another embodiment, the xenogenous immunoglobulin locus can be inherited through the male germ line by offspring. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
Definitions
• The terms “recombinant DNA technology,” “DNA cloning,” “molecular cloning,” or “gene cloning” refer to the process of transferring a DNA sequence into a cell or organism. The transfer of a DNA fragment can be from one organism to a self-replicating genetic element (e.g., bacterial plasmid) that permits a copy of any specific part of a DNA (or RNA) sequence to be selected among many others and produced in an unlimited amount. Plasmids and other types of cloning vectors such as artificial chromosomes can be used to copy genes and other pieces of chromosomes to generate enough identical material for further study. In addition to bacterial plasmids, which can carry up to 20 kb of foreign DNA, other cloning vectors include viruses, cosmids, and artificial chromosomes (e.g., bacteria artificial chromosomes (BACs) or yeast artificial chromosomes (YACs)). When the fragment of chromosomal DNA is ultimately joined with its cloning vector in the lab, it is called a “recombinant DNA molecule.” Shortly after the recombinant plasmid is introduced into suitable host cells, the newly inserted segment will be reproduced along with the host cell DNA.
• “Cosmids” are artificially constructed cloning vectors that carry up to 45 kb of foreign DNA. They can be packaged in lambda phage particles for infection into E. coli cells.
• As used herein, the term “mammal” (as in “genetically modified (or altered) mammal”) is meant to include any non-human mammal, including but not limited to pigs, sheep, goats, cattle (bovine), deer, mules, horses, monkeys, dogs, cats, rats, mice, birds, chickens, reptiles, fish, and insects. In one embodiment of the invention, genetically altered pigs and methods of production thereof are provided.
• The term “ungulate” refers to hoofed mammals. Artiodactyls are even-toed (cloven-hooved) ungulates, including antelopes, camels, cows, deer, goats, pigs, and sheep. Perissodactyls are odd toes ungulates, which include horses, zebras, rhinoceroses, and tapirs. The term ungulate as used herein refers to an adult, embryonic or fetal ungulate animal.
• As used herein, the terms “porcine”, “porcine animal”, “pig” and “swine” are generic terms referring to the same type of animal without regard to gender, size, or breed.
• A “homologous DNA sequence or homologous DNA” is a DNA sequence that is at least about 80%, 85%, 90%, 95%, 98% or 99% identical with a reference DNA sequence. A homologous sequence hybridizes under stringent conditions to the target sequence, stringent hybridization conditions include those that will allow hybridization occur if there is at least 85, at least 95% or 98% identity between the sequences.
• An “isogenic or substantially isogenic DNA sequence” is a DNA sequence that is identical to or nearly identical to a reference DNA sequence. The term “substantially isogenic” refers to DNA that is at least about 97-99% identical with the reference DNA sequence, or at least about 99.5-99.9% identical with the reference DNA sequence, and in certain uses 100% identical with the reference DNA sequence.
• “Homologous recombination” refers to the process of DNA recombination based on sequence homology.
• “Gene targeting” refers to homologous recombination between two DNA sequences, one of which is located on a chromosome and the other of which is not.
• “Non-homologous or random integration” refers to any process by which DNA is integrated into the genome that does not involve homologous recombination.
• A “selectable marker gene” is a gene, the expression of which allows cells containing the gene to be identified. A selectable marker can be one that allows a cell to proliferate on a medium that prevents or slows the growth of cells without the gene. Examples include antibiotic resistance genes and genes which allow an organism to grow on a selected metabolite. Alternatively, the gene can facilitate visual screening of transformants by conferring on cells a phenotype that is easily identified. Such an identifiable phenotype can be, for example, the production of luminescence or the production of a colored compound, or the production of a detectable change in the medium surrounding the cell.
• The term “contiguous” is used herein in its standard meaning, i.e., without interruption, or uninterrupted.
• “Stringent conditions” refers to conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C., or (2) employ during hybridization a denaturing agent such as, for example, formamide. One skilled in the art can determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. For example, stringency can generally be reduced by increasing the salt content present during hybridization and washing, reducing the temperature, or a combination thereof. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York, (1989).
I. Immunoglobulin Genes
• In one aspect of the present invention, a transgenic ungulate that lacks any expression of functional endogenous immunoglobulins is provided. In one embodiment, the ungulate can lack any expression of endogenous heavy and/or light chain immunoglobulins. The light chain immunoglobulin can be a kappa and/or lambda immunoglobulin. In additional embodiments, transgenic ungulates are provided that lack expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof. In one embodiment, the expression of functional endogenous immunoglobulins can be accomplished by genetic targeting of the endogenous immunoglobulin loci to prevent expression of the endogenous immunoglobulin. In one embodiment, the genetic targeting can be accomplished via homologous recombination. In another embodiment, the transgenic ungulate can be produced via nuclear transfer.
• In another aspect of the present invention, a method is provided to disrupt the expression of an ungulate immunoglobulin gene by (i) analyzing the germline configuration of the ungulate heavy chain, kappa light chain or lambda light chain genomic locus; (ii) determining the location of nucleotide sequences that flank the 5′ end and the 3′ end of at least one functional region of the locus; and (iii) transfecting a targeting construct containing the flanking sequence into a cell wherein, upon successful homologous recombination, at least one functional region of the immunoglobulin locus is disrupted thereby reducing or preventing the expression of the immunoglobulin gene.
• In one embodiment, the germline configuration of the porcine heavy chain locus is provided. The porcine heavy chain locus contains at least four variable regions, two diversity regions, six joining regions and five constant regions, for example, as illustrated in FIG. 1. In a specific embodiment, only one of the six joining regions, J6, is functional.
• In another embodiment, the germline configuration of the porcine kappa light chain locus is provided. The porcine kappa light chain locus contains at least six variable regions, six joining regions, one constant region and one enhancer region, for example, as illustrated in FIG. 2.
• In a further embodiment, the germline configuration of the porcine lambda light chain locus is provided.
• Isolated nucleotide sequences as depicted in Seq ID Nos 1-39 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to any one of Seq ID Nos 1-39 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of any one of Seq ID Nos 1-39 are provided. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1-39, as well as, nucleotides homologous thereto.
• Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (see, for example, Altschul, S. F. et al (1997) Nucleic Acids Res 25:3389-3402 and Karlin et al, (1900) Proc. Natl. Acad. Sci. USA 87, 2264-2268) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. See, for example, Altschul et aL, (1994) (Nature Genetics 6, 119-129). The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter (low co M'plexity) are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et aL, (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919), which is recommended for query sequences over 85 in length (nucleotide bases or amino acids).
Porcine Heavy Chain
• In another aspect of the present invention, novel genomic sequences encoding the heavy chain locus of ungulate immunoglobulin are provided. In one embodiment, an isolated nucleotide sequence encoding porcine heavy chain is provided that includes at least one variable region, two diversity regions, at least four joining regions and at least one constant region, such as the mu constant region, for example, as represented in Seq ID No. 29. In another embodiment, an isolated nucleotide sequence is provided that includes at least four joining regions and at least one constant region, such as the mu constant region, of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No. 4. In a further embodiment, nucleotide sequence is provided that includes 5′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No 1. Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in the 3′ region of Seq ID No 4. In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 1, 4 or 29 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 1, 4 or 29 are also provided. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1, 4 or 29, as well as, nucleotides homologous thereto.
• In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. In one embodiment, the nucleotide sequence contains at least 17, 20, 25 or 30 contiguous nucleotides of Seq ID No 4 or residues 1-9,070 of Seq ID No 29. In other embodiments, nucleotide sequences that contain at least 50, 100, 1,000, 2,500, 4,000, 4,500, 5,000, 7,000, 8,000, 8,500, 9,000, 10,000 or 15,000 contiguous nucleotides of Seq ID No. 29 are provided. In another embodiment, the nucleotide sequence contains residues 9,070-11039 of Seq ID No 29.
• In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 1, 4 or 29 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 1, 4 or 29 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1, 4 or 29, as well as, nucleotides homologous thereto.
• In one embodiment, an isolated nucleotide sequence encoding porcine heavy chain is provided that includes at least one variable region, two diversity regions, at least four joining regions and at least one constant region, such as the mu constant region, for example, as represented in Seq ID No. 29. In Seq ID No. 29, the Diversity region of heavy chain is represented, for example, by residues 1089-1099 (D(pseudo)), the Joining region of heavy chain is represented, for example, by residues 1887-3352 (for example: J(psuedo): 1887-1931, J(psuedo): 2364-2411, J(psuedo): 2756-2804, J (functional J): 3296-3352), the recombination signals are represented, for example, by residues 3001-3261 (Nonamer), 3292-3298 (Heptamer), the Constant Region is represented by the following residues: 3353-9070 (J to C mu intron), 5522-8700 (Switch region), 9071-9388 (Mu Exon 1), 9389-9469 (Mu Intron A), 9470-9802 (Mu Exon 2), 9830-10069 (Mu Intron B), 10070-10387 (Mu Exon 3), 10388-10517 (Mu Intron C), 10815-11052 (Mu Exon 4), 11034-11039 (Poly(A) signal).

• Seq ID No. 29

  tctagaagacgctggagagaggccagacttcctcggaacagctcaaagag

  ctctgtcaaagccagatcccatcacacgtgggcaccaataggccatgcca

  gcctccaagggccgaactgggttctccacggcgcacatgaagcctgcagc

  ctggcttatcctcttccgtggtgaagaggcaggcccgggactggacgagg

  ggctagcagggtgtggtaggcaccttgcgccccccaccccggcaggaacc

  agagaccctggggctgagagtgagcctccaaacaggatgccccacccttc

  aggccacctttcaatccagctacactccacctgccattctcctctgggca

  cagggcccagcccctggatcttggccttggctcgacttgcacccacgcgc

  acacacacacttcctaacgtgctgtccgctcacccctccccagcgtggtc

  catgggcagcacggcagtgcgcgtccggcggtagtgagtgcagaggtccc

  ttcccctcccccaggagccccaggggtgtgtgcagatctgggggctcctg

  tcccttacaccttcatgcccctcccctcatacccaccctccaggcgggag

  gcagcgagacctttgcccagggactcagccaacgggcacacgggaggcca

  gccctcagcagctggctcccaaagaggaggtgggaggtaggtccacagct

  gccacagagagaaaccctgacggaccccacaggggccacgccagccggaa

  ccagctccctcgtgggtgagcaatggccagggccccgccggccaccacgg

  ctggccttgcgccagctgagaactcacgtccagtgcagggagactcaaga

  cagcctgtgcacacagcctcggatctgctcccatttcaagcagaaaaagg

  aaaccgtgcaggcagccctcagcatttcaaggattgtagcagcggccaac

  tattcgtcggcagtggccgattagaatgaccgtggagaagggcggaaggg

  tctctcgtgggctctgcggccaacaggccctggctccacctgcccgctgc

  cagcccgaggggcttgggccgagccaggaaccacagtgctcaccgggacc

  acagtgactgaccaaactcccggccagagcagccccaggccagccgggct

  ctcgccctggaggactcaccatcagatgcacaagggggcgagtgtggaag

  agacgtgtcgcccgggccatttgggaaggcgaagggaccttccaggtgga

  caggaggtgggacgcactccaggcaagggactgggtccccaaggcctggg

  gaaggggtactggcttgggggttagcctggccagggaacggggagcgggg

  cggggggctgagcagggaggacctgacctcgtgggagcgaggcaagtcag

  gcttcaggcagcagccgcacatcccagaccaggaggctgaggcaggaggg

  gcttgcagcggggcgggggcctgcctggctccgggggctcctgggggacg

  ctggctcttgtttccgtgtcccgcagcacagggccagctcgctgggccta

  tgcttaccttgatgtctggggccggggcgtcagggtcgtcgtctcctcag

  gggagagtcccctgaggctacgctgggg*ggggactatggcagctccacc

  aggggcctggggaccaggggcctggaccaggctgcagcccggaggacggg

  cagggctctggctctccagcatctggccctcggaaatggcagaacccctg

  gcgggtgagcgagctgagagcgggtcagacagacaggggccggccggaaa

  ggagaagttgggggcagagcccgccaggggccaggcccaaggttctgtgt

  gccagggcctgggtgggcacattggtgtggccatggctacttagattcgt

  ggggccagggcatcctggtcaccgtctcctcaggtgagcctggtgtctga

  tgtccagctaggcgctggtgggccgcgggtgggcctgtctcaggctaggg

  caggggctgggatgtgtatttgtcaaggaggggcaacagggtgcagactg

  tgcccctggaaacttgaccactggggcaggggcgtcctggtcacgtctcc

  tcaggtaagacggccctgtgcccctctctcgcgggactggaaaaggaatt

  ttccaagattccttggtctgtgtggggccctctggggcccccgggggtgg

  ctcccctcctgcccagatggggcctcggcctgtggagcacgggctgggca

  cacagctcgagtctagggccacagaggcccgggctcagggctctgtgtgg

  cccggcgactggcagggggctcgggtttttggacaccccctaatgggggc

  cacagcactgtgaccatcttcacagctggggccgaggagtcgaggtcacc

  gtctcctcaggtgagtcctcgtcagccctctctcactctctggggggttt

  tgctgcattttgtgggggaaagaggatgcctgggtctcaggtctaaaggt

  ctagggccagcgccggggcccaggaaggggccgaggggccaggctcggct

  cggccaggagcagagcttccagacatctcgcctcctggcggctgcagtca

  ggcctttggccgggggggtctcagcaccaccaggcctcttggctcccgag

  gtccccggccccggctgcctcaccaggcaccgtgcgcggtgggcccgggc

  tcttggtcggccaccctttcttaactgggatccgggcttagttgtcgcaa

  tgtgacaacgggctcgaaagctggggccaggggaccctagtctacgacgc

  ctcgggtgggtgtcccgcacccctccccactttcacggcactcggcgaga

  cctggggagtcaggtgttggggacactttggaggtcaggaacgggagctg

  gggagagggctctgtcagcggggtccagagatgggccgccctccaaggac

  gccctgcgcggggacaagggcttcttggcctggcctggccgcttcacttg

  ggcgtcagggggggcttcccggggcaggcggtcagtcgaggcgggttgga

  attctgagtctgggttcggggttcggggttcggccttcatgaacagacag

  cccaggcgggccgttgtttggcccctgggggcctggttggaatgcgaggt

  ctcgggaagtcaggagggagcctggccagcagagggttcccagccctgcg

  gccgagggacctggagacgggcagggcattggccgtcgcagggccaggcc

  acaccccccaGGTTTTTGTggggcgagcctggagattgcacCACTGTGAT

  TACTATGCTATGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTC

  AGgtaagaacggccctccagggcctttaatttctgctctcgtctgtgggc

  ttttctgactctgatcctcgggaggcgtctgtgccccccccggggatgag

  gccggcttgccaggaggggtcagggaccaggagcctgtgggaagttctga

  cgggggctgcaggcgggaagggccccaccggggggcgagccccaggccgc

  tgggcggcaggagacccgtgagagtgcgccttgaggagggtgtctgcgga

  accacgaacgcccgccgggaagggcttgctgcaatgcggtcttcagacgg

  gaggcgtcttctgccctcaccgtctttcaagcccttgtgggtctgaaaga

  gccatgtcggagagagaagggacaggcctgtcccgacctggccgagagcg

  ggcagccccgggggagagcggggcgatcggcctgggctctgtgaggccag

  gtccaagggaggacgtgtggtcctcgtgacaggtgcacttgcgaaacctt

  agaagacggggtatgttggaagcggctcctgatgtttaagaaaagggaga

  ctgtaaagtgagcagagtcctcaagtgtgttaaggttttaaaggtcaaag

  tgttttaaacctttgtgactgcagttagcaagcgtgcggggagtgaatgg

  ggtgccagggtggccgagaggcagtacgagggccgtgccgtcctctaatt

  cagggcttagttttgcagaataaagtcggcctgttttctaaaagcattgg

  tggtgctgagctggtggaggaggccgcgggcagccctggccacctgcagc

  aggtggcaggaagcaggtcggccaagaggctattttaggaagccagaaaa

  cacggtcgatgaatttatagcttctggtttccaggaggtggttgggcatg

  gctttgcgcagcgccacagaaccgaaagtgcccactgagaaaaaacaact

  cctgcttaatttgcatttttctaaaagaagaaacagaggctgacggaaac

  tggaaagttcctgttttaactactcgaattgagttttcggtcttagctta

  tcaactgctcacttagattcattttcaaagtaaacgtttaagagccgagg

  cattcctatcctcttctaaggcgttattcctggaggctcattcaccgcca

  gcacctccgctgcctgcaggcattgctgtcaccgtcaccgtgacggcgcg

  cacgattttcagttggcccgcttcccctcgtgattaggacagacgcgggc

  actctggcccagccgtcttggctcagtatctgcaggcgtccgtctcggga

  cggagctcaggggaagagcgtgactccagttgaacgtgatagtcggtgcg

  ttgagaggagacccagtcgggtgtcgagtcagaaggggcccggggcccga

  ggccctgggcaggacggcccgtgccctgcatcacgggcccagcgtcctag

  aggcaggactctggtggagagtgtgagggtgcctggggcccctccggagc

  tggggccgtgcggtgcaggttgggctctcggcgcggtgttggctgtttct

  gcgggatttggaggaattcttccagtgatgggagtcgccagtgaccgggc

  accaggctggtaagagggaggccgccgtcgtggccagagcagctgggagg

  gttcggtaaaaggctcgcccgtttcctttaatgaggacttttcctggagg

  gcatttagtctagtcgggaccgttttcgactcgggaagagggatgcggag

  gagggcatgtgcccaggagccgaaggcgccgcggggagaagcccagggct

  ctcctgtccccacagaggcgacgccactgccgcagacagacagggccttt

  ccctctgatgacggcaaaggcgcctcggctcttgcggggtgctggggggg

  agtcgccccgaagccgctcacccagaggcctgaggggtgagactgaccga

  tgcctcttggccgggcctggggccggaccgagggggactccgtggaggca

  gggcgatggtggctgcgggagggaaccgaccctgggccgagcccggcttg

  gcgattcccgggcgagggccctcagccgaggcgagtgggtccggcggaac

  caccctttctggccagcgccacagggctctcgggactgtccggggcgacg

  ctgggctgcccgtggcaggccTGGGCTGACCTGGACTTCACCAGACAGAA

  CAGGGCTTTCAGGGCTGAGCTGAGCCAGGTTTAGCGAGGCCAAGTGGGGC

  TGAACCAGGCTCAACTGGCCTGAGCTGGGTTGAGCTGGGCTGACCTGGGC

  TGAGCTGAGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGAC

  TGGCTGAGCTGAGCTGGGTTGAGCTGAGCTGAGCTGGCCTGGGTTGAGCT

  GGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGTTGAGCTGGGTTGATCT

  GAGCTGAGCTGGGCTGAGCTGAGCTAGGCTGGGGTGAGCTGGGCTGAGCT

  GGTTTGAGTTGGGTTGAGCTGAGCTGAGCTGGGCTGTGCTGGCTGAGCTA

  GGCTGAGCTAGGCTAGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAGGCTG

  GGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCGTTGAGCTGGCTGG

  GCTGGATTGAGCTGGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGT

  TGAGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGGGTTGAGCTGTCC

  TGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTCAGC

  AGAGCTGGGTTGGGCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCTGGCC

  TGGGTTGAGCTGGGCTGAGCTGAGCTGGGCTGAGCTGGCCTGTGTTGAGC

  TGGGCTGGGTTGAGCTGGGCTGAGCTGGATTGAGCTGGGTTGAGCTGAGC

  TGGGCTGGGCTGTGCTGACTGAGCTGGGCTGAGCTAGGCTGGGGTGAGCT

  GGGCTGAGCTGATCCGAGCTAGGCTGGGCTGGTTTGGGCTGAGCTGAGCT

  GAGCTAGGCTGGATTGATCTGGCTGAGCTGGGTTGAGCTGAGCTGGGCTG

  AGCTGGTCTGAGCTGGCCTGGGTCGAGCTGAGCTGGACTGGTTTGAGCTG

  GGTCGATCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCTG

  AGCTGGGTTGAGCTGGGCTGAGCTGAGGGCTGGGGTGAGCTGGGCTGAAC

  TAGCCTAGCTAGGTTGGGCTGAGCTGGGCTGGTTTGGGCTGAGCTGAGCT

  GAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCAGGCCTGGGGTG

  AGCTGGGCTAGGTGGAGCTGAGCTGGGTCGAGCTGAGTTGGGCTGAGCTG

  GCCTGGGTTGAGGTAGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTG

  GCTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGTTGA

  GCTGGGCTCGGTTGAGCTGGGCTGAGCTGAGCCGACCTAGGCTGGGATGA

  GCTGGGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGG

  CTGAGCTGGGCCTGGAGCCTGGCCTGGGGTGAGCTGGGCTGAGCTGCGCT

  GAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAAGCTG

  GGCCGAGCTGGCCTGGGATGAGCTGGGCCGGTTTGGGCTGAGCTGAGCTG

  AGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCTGGCCTGGGGTGA

  GCTGGGCTGAGCTAAGCTGAGCTGGGCTGGTTTGGGCTGAGCTGGCTGAG

  CTGGGTCCTGCTGAGCTGGGCTGAGCTGACCAGGGGTGAGCTGGGCTGAG

  TTAGGCTGGGCTCAGCTAGGCTGGGTTGATCTGGCAGGGCTGGTTTGCGC

  TGGGTCAAGCTCCCGGGAGATGGCCTGGGATGAGCTGGGCTGGTTTGGGC

  TGAGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCT

  GAGCTGGCCTGGGGTGAGCTGGGCTGGGTGGAGCTGAGCTGGGCTGAACT

  GGGCTAAGCTGGCTGAGCTGGATCGAGCTGAGCTGGGCTGAGCTGGCCTG

  GGGTTAGCTGGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGCTGG

  GCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGTTGAGCTGG

  GCTGGGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGGCTGGGCTGAGCTGA

  GCTAGGCTGCATTGAGCTGGCTGGGATGGATTGAGCTGGCTGAGCTGGCT

  GAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTG

  AGCTGAGCTGGGCTGAGCTGGGCTCAGCAGAGCTGGGTTGAGCTGAGCTG

  GGTTGAGCTGGGGTGAGCTGGGCTGAGCAGAGCTGGGTTGAGCTGAGCTG

  GGTTGAGCTGGGCTCGAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCT

  GGGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTGAGCT

  AGCTGGGCTCAGCTAGGCTGGGTTGAGCTGAGCTGGGCTGAACTGGGCTG

  AGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCTGGGCTGAGCAGAGCTG

  GGCTGAGCAGAGCTGGGTTGGTCTGAGCTGGGTTGAGCTGGGCTGAGCTG

  GGCTGAGCAGAGTTGGGTTGAGCTGAGCTGGGTTCAGCTGGGCTGAGCTA

  GGCTGGGTTGAGCTGGGTTGAGTTGGGCTGAGCTGGGCTGGGTTGAGCGG

  AGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCGGAACTGGGTTGATCTG

  AATTGAGCTGGGCTGAGCCGGGCTGAGCCGGGCTGAGCTGGGCTAGGTTG

  AGCTTGGGTGAGCTTGCCTCAGCTGGTCTGAGCTAGGTTGGGTGGAGCTA

  GGCTGGATTGAGCTGGGCTGAGCTGAGCTGATCTGGCCTCAGCTGGGCTG

  AGGTAGGCTGAACTGGGCTGTGCTGGGCTGAGCTGAGCTGAGCCAGTTTG

  AGCTGGGTTGAGCTGGGCTGAGCTGGGCTGTGTTGATCTTTCCTGAACTG

  GGCTGAGCTGGGCTGAGCTGGCCTAGCTGGATTGAACGGGGGTAAGCTGG

  GCCAGGCTGGACTGGGCTGAGCTGAGCTAGGCTGAGCTGAGTTGAATTGG

  GTTAAGCTGGGCTGAGATGGGCTGAGCTGGGCTGAGCTGGGTTGAGCCAG

  GTCGGACTGGGTTACCCTGGGCCACACTGGGCTGAGCTGGGCGGAGCTCG

  attaacctggtcaggctgagtcgggtccagcagacatgcgctggccaggc

  tggcttgacctggacacgttcgatgagctgccttgggatggttcacctca

  gctgagccaggtggctccagctgggctgagctggtgaccctgggtgacct

  cggtgaccaggttgtcctgagtccgggccaagccgaggctgcatcagact

  cgccagacccaaggcctgggccccggctggcaagccaggggcggtgaagg

  ctgggctggcaggactgtcccggaaggaggtgcacgtggagccgcccgga

  ccccgaccggcaggacctggaaagacgcctctcactcccctttctcttct

  gtcccctctcgggtcctcagAGAGCCAGTCTGCCCCGAATCTCTACCCCC

  TCGTCTCCTGCGTCAGCCCCCCGTCCGATGAGAGCCTGGTGGCCCTGGGC

  TGCCTGGCCCGGGACTTCCTGCCCAGCTCCGTCACCTTCTCCTGGAACTA

  CAAGAACAGCAGCAAGGTCAGCAGCCAGAACATCCAGGACTTCCCGTCCG

  TCCTGAGAGGCGGCAAGTACTTGGCCTCCTCCCGGGTGCTCCTACCCTCT

  GTGAGCATCCCCCAGGACCCAGAGGCCTTCCTGGTGTGCGAGGTCCAGCA

  CCCCAGTGGCACCAAGTCCGTGTCCATCTCTGGGCCAGgtgagctgggct

  ccccctgtggctgtggcgggggcggggccgggtgccgccggcacagtgac

  gccccgttcctgcctgcagTCGTAGAGGAGCAGCCCCCCGTCTTGAACAT

  CTTCGTCCCCACCCGGGAGTCCTTCTCCAGTACTCCCCAGCGCACGTCCA

  AGCTCATCTGCCAGGCCTCAGACTTCAGCCCCAAGCAGATCTCCATGGCC

  TGGTTCCGTGATGGGAAACGGGTGGTGTCTGGCGTCAGCACAGGCCCCGT

  GGAGACCCTACAGTCCAGTCCGGTGACCTACAGGCTCCACAGCATGCTGA

  CCGTCACGGAGTCCGAGTGGCTCAGCCAGAGCGTCTTCACCTGCCAGGTG

  GAGCACAAAGGGCTGAACTACGAGAAGAACGCGTCCTCTCTGTGCACCTC

  CAgtgagtgcagcccctcgggccgggcggcggggcggcgggagccacaca

  cacaccagctgctccctgagccttggcttccgggagtggccaaggcgggg

  aggggctgtgcagggcagctggagggcactgtcagctggggcccagcacc

  ccctcaccccggcagggcccgggctccgaggggccccgcagtcgcaggcc

  ctgctcttgggggaagccctacttggccccttcagggcgctgacgctccc

  cccacccacccccgcctagATCCCAACTCTCCCATCACCGTCTTCGCCAT

  CGCCCCCTCCTTCGCTGGCATCTTCCTCACCAAGTCGGCCAAGCTTTCCT

  GCCTGGTCACGGGCCTCGTCACCAGGGAGAGCCTCAACATCTCCTGGACC

  CGCCAGGACGGCGAGGTTCTGAAGACCAGTATCGTCTTCTCTGAGATCTA

  CGCCAACGGCACCTTCGGCGCCAGGGGCGAAGCCTCCGTCTGCGTGGAGG

  ACTGGGAGTCGGGCGACAGGTTCACGTGCACGGTGACCCACACGGACCTG

  CCCTCGCCGCTGAAGCAGAGCGTCTCCAAGCCCAGAGgtaggccctgccc

  tgcccctgcctccgcccggcctgtgccttggccgccggggcgggagccga

  gcctggccgaggagcgccctcggccccccgcggtcccgacccacacccct

  cctgctctcctccccagGGATCGCCAGGCACATGCCGTCCGTGTACGTGC

  TGCCGCCGGCCCCGGAGGAGCTGAGCCTGCAGGAGTGGGCCTCGGTCACC

  TGCCTGGTGAAGGGCTTCTCCCCGGCGGACGTGTTCGTGCAGTGGCTGCA

  GAAGGGGGAGCCCGTGTCCGCCGACAAGTACGTGACCAGCGCGCCGGTGC

  CCGAGCCCGAGCCCAAGGCCCCCGCCTCCTACTTCGTGCAGAGCGTCCTG

  ACGGTGAGCGCCAAGGACTGGAGCGACGGGGAGACCTACACCTGCGTCGT

  GGGCCACGAGGCCCTGCCCCACACGGTGACCGAGAGGACCGTGGACAAGT

  CCACCGGTAAACCCACCCTGTACAACGTCTCCCTGGTCCTGTCCGACACG

  GCCAGCACCTGCTACTGACCCCCTGGCTGCCCGCCGCGGCCGGGGCCAGA

  GCCCCCGGGCGACCATCGCTCTGTGTGGGCCTGTGTGCAACCCGACCCTG

  TCGGGGTGAGCGGTCGCATTTCTGAAAATTAGAaataaaAGATCTCGTGC

  CG

  Seq ID No. 1

  TCTAgAAGACGCTGGAGAGAGGCCagACTTCCTCGGAACAGCTCAAAGAG

  CTCTGTCAAAGCCAGATCCCATCACACGTGGGCACCAATAGGCCATGCCA

  GCCTCCAAGGGCCGAACTGGGTTCTCCACGGCGCACATGAAGCCTGCAGC

  CTGGCTTATCCTCTTCCGTGGTGAAGAGGCAGGCCCGGGACTGGACGAGG

  GGCTAGCAGGGTGTGGTAGGCACCTTGCGCCCCCCACCCCGGCAGGAACC

  AGAGACCCTGGGGCTGAGAGTGAGCCTCCAAACAGGATGCCCCACCCTTC

  AGGCCACCTTTCAATCCAGCTACACTCCACCTGCCATTCTCCTCTGGGCA

  CAGGGCCCAGCCCCTGGATCTTGGCCTTGGCTCGACTTGCACCCACGCGC

  ACACACACACTTCCTAACGTGCTGTCCGCTCACCCCTCCCCAGCGTGGTC

  CATGGGCAGCACGGCAGTGCGCGTCCGGCGGTAGTGAGTGCAGAGGTCCC

  TTCCCCTCCCCCAGGAGCCCCAGGGGTGTGTGCAGATCTGGGGGCTCCTG

  TCCCTTACACCTTCATGCCCCTCCCCTCATACCCACCCTCCAGGCGGGAG

  GCAGCGAGACCTTTGCCCAGGGACTCAGCCAACGGGCACACGGGAGGCCA

  GCCCTCAGCAGCTGGG

  Seq ID No. 4

  GGCCAGACTTCCTCGGAACAGCTCAAAGAGCTCTGTCAAAGCCAGATCCC

  ATCACACGTGGGCACCAATAGGCCATGCCAGCCTCCAAGGGCCGAACTGG

  GTTCTCCACGGCGCACATGAAGCCTGCAGCCTGGCTTATCCTCTTCCGTG

  GTGAAGAGGCAGGCCCGGGACTGGACGAGGGGCTAGCAGGGTGTGGTAGG

  CACCTTGCGCCCCCCACCCCGGCAGGAACCAGAGACCCTGGGGCTGAGAG

  TGAGCCTCCAAACAGGATGCCCCACCCTTCAGGCCACCTTTCAATCCAGC

  TACACTCCACCTGCCATTCTCCTCTGGGCACAGGGCCCAGCCCCTGGATC

  TTGGCCTTGGCTCGACTTGCACCCACGCGCACACACACACTTCCTAACGT

  GCTGTCCGCTCACCCCTCCCCAGCGTGGTCCATGGGCAGCACGGCAGTGC

  GCGTCCGGCGGTAGTGAGTGCAGAGGTCCCTTCCCCTCCCCCAGGAGCCC

  CAGGGGTGTGTGCAGATCTGGGGGCTCCTGTCCCTTACACCTTCATGCCC

  CTCCCCTCATACCCACCCTCCAGGCGGGAGGCAGCGAGACCTTTGCCCAG

  GGACTCAGCCAACGGGCACACGGGAGGCCAGCCCTCAGCAGCTGGCTCCC

  AAAGAGGAGGTGGGAGGTAGGTCCACAGCTGCCACAGAGAGAAACCCTGA

  CGGACCCCACAGGGGCCACGCCAGCCGGAACCAGCTCCCTCGTGGGTGAG

  CAATGGCCAGGGCCCCGCCGGCCACCACGGCTGGCCTTGCGCCAGCTGAG

  AACTCACGTCCAGTGCAGGGAGACTCAAGACAGCCTGTGCACACAGCCTC

  GGATCTGCTCCCATTTCAAGCAGAAAAAGGAAACCGTGCAGGCAGCCCTC

  AGCATTTCAAGGATTGTAGCAGCGGCCAACTATTCGTCGGCAGTGGCCGA

  TTAGAATGACCGTGGAGAAGGGCGGAAGGGTCTCTCGTGGGCTCTGCGGC

  CAACAGGCCCTGGCTCCACCTGCCCGCTGCCAGCCCGAGGGGCTTGGGCC

  GAGCCAGGAACCACAGTGCTCACCGGGACCACAGTGACTGACCAAACTCC

  CGGCCAGAGCAGCCCCAGGCCAGCCGGGCTCTCGCCCTGGAGGACTCACC

  ATCAGATGCACAAGGGGGCGAGTGTGGAAGAGACGTGTCGCCCGGGCCAT

  TTGGGAAGGCGAAGGGACCTTCCAGGTGGACAGGAGGTGGGACGCACTCC

  AGGCAAGGGACTGGGTCCCCAAGGCCTGGGGAAGGGGTACTGGCTTGGGG

  GTTAGCCTGGCCAGGGAACGGGGAGCGGGGCGGGGGGCTGAGCAGGGAGG

  ACCTGACCTCGTGGGAGCGAGGCAAGTCAGGCTTCAGGCAGCAGCCGCAC

  ATCCCAGACCAGGAGGCTGAGGCAGGAGGGGCTTGCAGCGGGGCGGGGGC

  CTGCCTGGCTCCGGGGGCTCCTGGGGGACGCTGGCTCTTGTTTCCGTGTC

  CCGCAGCACAGGGCCAGCTCGCTGGGCCTATGCTTACCTTGATGTCTGGG

  GCCGGGGCGTCAGGGTCGTCGTCTCCTCAGGGGAGAGTCCCCTGAGGCTA

  CGCTGGGG*GGGGACTATGGCAGCTCCACCAGGGGCCTGGGGACCAGGGG

  CCTGGACCAGGCTGCAGCCCGGAGGACGGGCAGGGCTCTGGCTCTCCAGC

  ATCTGGCCCTCGGAAATGGCAGAACCCCTGGCGGGTGAGCGAGCTGAGAG

  CGGGTCAGACAGACAGGGGCCGGCCGGAAAGGAGAAGTTGGGGGCAGAGC

  CCGCCAGGGGCCAGGCCCAAGGTTCTGTGTGCCAGGGCCTGGGTGGGCAC

  ATTGGTGTGGCCATGGCTACTTAGATTCGTGGGGCCAGGGCATCCTGGTC

  ACCGTCTCCTCAGGTGAGCCTGGTGTCTGATGTCCAGCTAGGCGCTGGTG

  GGCCGCGGGTGGGCCTGTCTCAGGCTAGGGCAGGGGCTGGGATGTGTATT

  TGTCAAGGAGGGGCAACAGGGTGCAGACTGTGCCCCTGGAAACTTGACCA

  CTGGGGCAGGGGCGTCCTGGTCACGTCTCCTCAGGTAAGACGGCCCTGTG

  CCCCTCTCTCGCGGGACTGGAAAAGGAATTTTCCAAGATTCCTTGGTCTG

  TGTGGGGCCCTCTGGGGCCCCCGGGGGTGGCTCCCCTCCTGCCCAGATGG

  GGCCTCGGCCTGTGGAGCACGGGCTGGGCACACAGCTCGAGTCTAGGGCC

  ACAGAGGCCCGGGCTCAGGGCTCTGTGTGGCCCGGCGACTGGCAGGGGGC

  TCGGGTTTTTGGACACCCCCTAATGGGGGCCACAGCACTGTGACCATCTT

  CACAGCTGGGGCCGAGGAGTCGAGGTCACCGTCTCCTCAGGTGAGTCCTC

  GTCAGCCCTCTCTCACTCTCTGGGGGGTTTTGCTGCATTTTGTGGGGGAA

  AGAGGATGCCTGGGTCTCAGGTCTAAAGGTCTAGGGCCAGCGCCGGGGCC

  CAGGAAGGGGCCGAGGGGCCAGGCTCGGCTCGGCCAGGAGCAGAGCTTCC

  AGACATCTCGCCTCCTGGCGGCTGCAGTCAGGCCTTTGGCCGGGGGGGTC

  TCAGCACCACCAGGCCTCTTGGCTCCCGAGGTCCCCGGCCCCGGCTGCCT

  CACCAGGCACCGTGCGCGGTGGGCCCGGGCTCTTGGTCGGCCACCCTTTC

  TTAACTGGGATCCGGGCTTAGTTGTCGCAATGTGACAACGGGCTCGAAAG

  CTGGGGCCAGGGGACCCTAGT*TACGACGCCTCGGGTGGGTGTCCCGCAC

  CCCTCCCCACTTTCACGGCACTCGGCGAGACCTGGGGAGTCAGGTGTTGG

  GGACACTTTGGAGGTCAGGAACGGGAGCTGGGGAGAGGGCTCTGTCAGCG

  GGGTCCAGAGATGGGCCGCCCTCCAAGGACGCCCTGCGCGGGGACAAGGG

  CTTCTTGGCCTGGCCTGGCCGCTTCACTTGGGCGTCAGGGGGGGCTTCCC

  GGGGCAGGCGGTCAGTCGAGGCGGGTTGGAATTCTGAGTCTGGGTTCGGG

  GTTCGGGGTTCGGCCTTCATGAACAGACAGCCCAGGCGGGCCGTTGTTTG

  GCCCCTGGGGGCCTGGTTGGAATGCGAGGTCTCGGGAAGTCAGGAGGGAG

  CCTGGCCAGCAGAGGGTTCCCAGCCCTGCGGCCGAGGGACCTGGAGACGG

  GCAGGGCATTGGCCGTCGCAGGGCCAGGCCACACCCCCCAGGTTTTTGTG

  GGGCGAGCCTGGAGATTGCACCACTGTGATTACTATGCTATGGATCTCTG

  GGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAGGTAAGAACGGCCCTCCAG

  GGCCTTTAATTTCTGCTCTCGTCTGTGGGCTTTTCTGACTCTGATCCTCG

  GGAGGCGTCTGTGCCCCCCCCGGGGATGAGGCCGGCTTGCCAGGAGGGGT

  CAGGGACCAGGAGCCTGTGGGAAGTTCTGACGGGGGCTGCAGGCGGGAAG

  GGCCCCACCGGGGGGCGAGCCCCAGGCCGCTGGGCGGCAGGAGACCCGTG

  AGAGTGCGCCTTGAGGAGGGTGTCTGCGGAACCACGAACGCCCGCCGGGA

  AGGGCTTGCTGCAATGCGGTCTTCAGACGGGAGGCGTCTTCTGCCCTCAC

  CGTCTTTCAAGCCCTTGTGGGTCTGAAAGAGCCATGTCGGAGAGAGAAGG

  GACAGGCCTGTCCCGACCTGGCCGAGAGCGGGCAGCCCCGGGGGAGAGCG

  GGGCGATCGGCCTGGGCTCTGTGAGGCCAGGTCCAAGGGAGGACGTGTGG

  TCCTCGTGACAGGTGCACTTGCGAAACCTTAGAAGACGGGGTATGTTGGA

  AGCGGCTCCTGATGTTTAAGAAAAGGGAGACTGTAAAGTGAGCAGAGTCC

  TCAAGTGTGTTAAGGTTTTAAAGGTCAAAGTGTTTTAAACCTTTGTGACT

  GCAGTTAGCAAGCGTGCGGGGAGTGAATGGGGTGCCAGGGTGGCCGAGAG

  GCAGTACGAGGGCCGTGCCGTCCTCTAATTCAGGGCTTAGTTTTGCAGAA

  TAAAGTCGGCCTGTTTTCTAAAAGCATTGGTGGTGCTGAGCTGGTGGAGG

  AGGCCGCGGGCAGCCCTGGCCACCTGCAGCAGGTGGCAGGAAGCAGGTCG

  GCCAAGAGGCTATTTTAGGAAGCCAGAAAACACGGTCGATGAATTTATAG

  CTTCTGGTTTCCAGGAGGTGGTTGGGCATGGCTTTGCGCAGCGCCACAGA

  ACCGAAAGTGCCCACTGAGAAAAAACAACTCCTGCTTAATTTGCATTTTT

  CTAAAAGAAGAAACAGAGGCTGACGGAAACTGGAAAGTTCCTGTTTTAAC

  TACTCGAATTGAGTTTTCGGTCTTAGCTTATCAACTGCTCACTTAGATTC

  ATTTTCAAAGTAAACGTTTAAGAGCCGAGGCATTCCTATCCTCTTCTAAG

  GCGTTATTCCTGGAGGCTCATTCACCGCCAGCACCTCCGCTGCCTGCAGG

  CATTGCTGTCACCGTCACCGTGACGGCGCGCACGATTTTCAGTTGGCCCG

  CTTCCCCTCGTGATTAGGACAGACGCGGGCACTCTGGCCCAGCCGTCTTG

  GCTCAGTATCTGCAGGCGTCCGTCTCGGGACGGAGCTCAGGGGAAGAGCG

  TGACTCCAGTTGAACGTGATAGTCGGTGCGTTGAGAGGAGACCCAGTCGG

  GTGTCGAGTCAGAAGGGGCCCGGGGCCCGAGGCCCTGGGCAGGACGGCCC

  GTGCCCTGCATCACGGGCCCAGCGTCCTAGAGGCAGGACTCTGGTGGAGA

  GTGTGAGGGTGCCTGGGGCCCCTCCGGAGCTGGGGCCGTGCGGTGCAGGT

  TGGGCTCTCGGCGCGGTGTTGGCTGTTTCTGCGGGATTTGGAGGAATTCT

  TCCAGTGATGGGAGTCGCCAGTGACCGGGCACCAGGCTGGTAAGAGGGAG

  GCCGCCGTCGTGGCCAGAGCAGCTGGGAGGGTTCGGTAAAAGGCTCGCCC

  GTTTCCTTTAATGAGGACTTTTCCTGGAGGGCATTTAGTCTAGTCGGGAC

  CGTTTTCGACTCGGGAAGAGGGATGCGGAGGAGGGCATGTGCCCAGGAGC

  CGAAGGCGCCGCGGGGAGAAGCCCAGGGCTCTCCTGTCCCCACAGAGGCG

  ACGCCACTGCCGCAGACAGACAGGGCCTTTCCCTCTGATGACGGCAAAGG

  CGCCTCGGCTCTTGCGGGGTGCTGGGGGGGAGTCGCCCCGAAGCCGCTCA

  CCCAGAGGCCTGAGGGGTGAGACTGACCGATGCCTCTTGGCCGGGCCTGG

  GGCCGGACCGAGGGGGACTCCGTGGAGGCAGGGCGATGGTGGCTGCGGGA

  GGGAACCGACCCTGGGCCGAGCCCGGCTTGGCGATTCCCGGGCGAGGGCC

  CTCAGCCGAGGCGAGTGGGTCCGGCGGAACCACCCTTTCTGGCCAGCGCC

  ACAGGGCTCTCGGGACTGTCCGGGGCGACGCTGGGCTGCCCGTGGCAGGC

  CTGGGCTGACCTGGACTTCACCAGACAGAACAGGGCTTTCAGGGCTGAGC

  TGAGCCAGGTTTAGCGAGGCCAAGTGGGGCTGAACCAGGCTCAACTGGCC

  TGAGCTGGGTTGAGCTGGGCTGACCTGGGCTGAGCTGAGCTGGGCTGGGC

  TGGGCTGGGCTGGGCTGGGCTGGGCTGGACTGGCTGAGCTGAGCTGGGTT

  GAGCTGAGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCT

  GGGTTGAGCTGGGTTGAGCTGGGTTGATCTGAGCTGAGCTGGGCTGAGCT

  GAGCTAGGCTGGGGTGAGCTGGGCTGAGCTGGTTTGAGTTGGGTTGAGCT

  GAGCTGAGCTGGGCTGTGCTGGCTGAGCTAGGCTGAGCTAGGCTAGGTTG

  AGCTGGGCTGGGCTGAGCTGAGCTAGGCTGGGCTGATTTGGGCTGAGCTG

  AGCTGAGCTAGGCTGCGTTGAGCTGGCTGGGCTGGATTGAGCTGGCTGAG

  CTGGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGAGCTGGACTGGTT

  TGAGCTGGGTCGATCTGGGTTGAGCTGTCCTGGGTTGAGCTGGGCTGGGT

  TGAGCTGAGCTGGGTTGAGCTGGGCTCAGCAGAGCTGGGTTGGGCTGAGC

  TGGGTTGAGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGAGC

  TGAGCTGGGCTGAGCTGGCCTGTGTTGAGCTGGGCTGGGTTGAGCTGGGC

  TGAGCTGGATTGAGCTGGGTTGAGCTGAGCTGGGCTGGGCTGTGCTGACT

  GAGCTGGGCTGAGCTAGGCTGGGGTGAGCTGGGCTGAGCTGATCCGAGCT

  AGGCTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTAGGCTGGATTGATCT

  GGCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCTGGTCTGAGCTGGCCTG

  GGTCGAGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGGGCTGAGCTG

  GCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTG

  AGCTGAGGGCTGGGGTGAGCTGGGCTGAACTAGCCTAGCTAGGTTGGGCT

  GAGCTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCA

  GGCTGAGCTGGGCTGAGCAGGCCTGGGGTGAGCTGGGCTAGGTGGAGCTG

  AGCTGGGTCGAGCTGAGTTGGGCTGAGCTGGCCTGGGTTGAGGTAGGCTG

  AGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGG

  GTCAAGCTGGGCCGAGCTGGCCTGGGTTGAGCTGGGCTCGGTTGAGCTGG

  GCTGAGCTGAGCCGACCTAGGCTGGGATGAGCTGGGCTGATTTGGGCTGA

  GCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCCTGGAGCCT

  GGCCTGGGGTGAGCTGGGCTGAGCTGCGCTGAGCTAGGCTGGGTTGAGCT

  GGCTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGATG

  AGCTGGGCCGGTTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAG

  GCTGAGCTGGGCTGAGCTGGCCTGGGGTGAGCTGGGCTGAGCTAAGCTGA

  GCTGGGCTGGTTTGGGCTGAGCTGGCTGAGCTGGGTCCTGCTGAGCTGGG

  CTGAGCTGACCAGGGGTGAGCTGGGCTGAGTTAGGCTGGGCTCAGCTAGG

  CTGGGTTGATCTGGCAGGGCTGGTTTGCGCTGGGTCAAGCTCCCGGGAGA

  TGGCCTGGGATGAGCTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTGAGC

  TAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCTGGCCTGGGGTGAGCT

  GGGCTGGGTGGAGCTGAGCTGGGCTGAACTGGGCTAAGCTGGCTGAGCTG

  GATCGAGCTGAGCTGGGCTGAGCTGGCCTGGGGTTAGCTGGGCTGAGCTG

  AGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAA

  GCTGGGCCGAGCTGGCCTGGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAG

  GCTGGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAGGCTGCATTGAGCTGG

  CTGGGATGGATTGAGCTGGCTGAGCTGGCTGAGCTGGCTGAGCTGGGCTG

  AGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGCTGAGCTG

  GGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGGTGAGCTG

  GGCTGAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTCGAGCA

  GAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTCAGCAGAGCTGGGTT

  GAGCTGAGCTGGGTTGAGCTGGGCTGAGCTAGCTGGGCTCAGCTAGGCTG

  GGTTGAGCTGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAACTGGGCTG

  AGCTGGGCTGAGCTGGGCTGAGCAGAGCTGGGCTGAGCAGAGCTGGGTTG

  GTCTGAGCTGGGTTGAGCTGGGCTGAGCTGGGCTGAGCAGAGTTGGGTTG

  AGCTGAGCTGGGTTCAGCTGGGCTGAGCTAGGCTGGGTTGAGCTGGGTTG

  AGTTGGGCTGAGCTGGGCTGGGTTGAGCGGAGCTGGGCTGAACTGGGCTG

  AGCTGGGCTGAGCGGAACTGGGTTGATCTGAATTGAGCTGGGCTGAGCCG

  GGCTGAGCCGGGCTGAGCTGGGCTAGGTTGAGCTTGGGTGAGCTTGCCTC

  AGCTGGTCTGAGCTAGGTTGGGTGGAGCTAGGCTGGATTGAGCTGGGCTG

  AGCTGAGCTGATCTGGCCTCAGCTGGGCTGAGGTAGGCTGAACTGGGCTG

  TGCTGGGCTGAGCTGAGCTGAGCCAGTTTGAGCTGGGTTGAGCTGGGCTG

  AGCTGGGCTGTGTTGATCTTTCCTGAACTGGGCTGAGCTGGGCTGAGCTG

  GCCTAGCTGGATTGAACGGGGGTAAGCTGGGCCAGGCTGGACTGGGCTGA

  GCTGAGCTAGGCTGAGCTGAGTTGAATTGGGTTAAGCTGGGCTGAGATGG

  GCTGAGCTGGGCTGAGCTGGGTTGAGCCAGGTCGGACTGGGTTACCCTGG

  GCCACACTGGGCTGAGCTGGGCGGAGCTCGATTAACCTGGTCAGGCTGAG

  TCGGGTCCAGCAGACATGCGCTGGCCAGGCTGGCTTGACCTGGACACGTT

  CGATGAGCTGCCTTGGGATGGTTCACCTCAGCTGAGCCAGGTGGCTCCAG

  CTGGGCTGAGCTGGTGACCCTGGGTGACCTCGGTGACCAGGTTGTCCTGA

  GTCCGGGCCAAGCCGAGGCTGCATCAGACTCGCCAGACCCAAGGCCTGGG

  CCCCGGCTGGCAAGCCAGGGGCGGTGAAGGCTGGGCTGGCAGGACTGTCC

  CGGAAGGAGGTGCACGTGGAGCCGCCCGGACCCCGACCGGCAGGACCTGG

  AAAGACGCCTCTCACTCCCCTTTCTCTTCTGTCCCCTCTCGGGTCCTCAG

  AGAGCCAGTCTGCCCCGAATCTCTACCCCCTCGTCTCCTGCGTCAGCCCC

  CCGTCCGATGAGAGCCTGGTGGCCCTGGGCTGCCTGGCCCGGGACTTCCT

  GCCCAGCTCCGTCACCTTCTCCTGGAA

Porcine Kappa Light Chain
• In another embodiment, novel genomic sequences encoding the kappa light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate kappa light chain regions. In one embodiment, nucleic acid sequence is provided that encodes the porcine kappa light chain locus. In another embodiment, the nucleic acid sequence can contain at least one joining region, one constant region and/or one enhancer region of kappa light chain. In a further embodiment, the nucleotide sequence can include at least five joining regions, one constant region and one enhancer region, for example, as represented in Seq ID No. 30. In a further embodiment, an isolated nucleotide sequence is provided that contains at least one, at least two, at least three, at least four or five joining regions and 3′ flanking sequence to the joining region of porcine genomic kappa light chain, for example, as represented in Seq ID No 12. In another embodiment, an isolated nucleotide sequence of porcine genomic kappa light chain is provided that contains 5′ flanking sequence to the first joining region, for example, as represented in Seq ID No. 25. In a further embodiment, an isolated nucleotide sequence is provided that contains 3′ flanking sequence to the constant region and, optionally, the 5′ portion of the enhancer region, of porcine genomic kappa light chain, for example, as represented in Seq ID Nos. 15, 16 and/or 19.
• In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 30, 12, 25, 15, 16 or 19 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. In other embodiments, nucleotide sequences that contain at least 50, 100, 1,000, 2,500, 5,000, 7,000, 8,000, 8,500, 9,000, 10,000 or 15,000 contiguous nucleotides of Seq ID No. 30 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 30, 12, 25, 15, 16 or 19, as well as, nucleotides homologous thereto.
• In one embodiment, an isolated nucleotide sequence encoding kappa light chain is provided that includes at least five joining regions, one constant region and one enhancer region, for example, as represented in Seq ID No. 30. In Seq ID No. 30, the coding region of kappa light chain is represented, for example by residues 1-549 and 10026-10549, whereas the intronic sequence is represented, for example, by residues 550-10025, the Joining region of kappa light chain is represented, for example, by residues 5822-7207 (for example, J1:5822-5859, J2:6180-6218, J3:6486-6523, J4:6826-6863, J5:7170-7207), the Constant Region is represented by the following residues: 10026-10549 (C exon) and 10026-10354 (C coding), 10524-10529 (Poly(A) signal) and 11160-11264 (SINE element).

• Seq ID No 30

  GCGTCCGAAGTCAAAAATATCTGCAGCCTTCATGTATTCATAGAAACAAG

  GAATGTCTACATTTTCCAAAGTGGGACCAGAATCTTGGGTCATGTCTAAG

  GCATGTGCATTTGCACATGGTAGGCAAAGGACTTTGCTTCTCCCAGCACA

  TCTTTCTGCAGAGATCCATGGAAACAAGACTCAACTCCAAAGCAGCAAAG

  AAGCAGCAAGTTCTCAAGTGATCTCCTCTGACTCCCTCCTCCCAGGCTAA

  TGAAGCCATGTTGCCCCTGGGGGATTAAGGGCAGGTGTCCATTGTGGCAC

  CCAGCCCGAAGACAAGCAATTTGATCAGGTTCTGAGCACTCCTGAATGTG

  GACTCTGGAATTTTCTCCTCACCTTGTGGCATATCAGCTTAAGTCAAGTA

  CAAGTGACAAACAACATAATCCTAAGAAGAGAGGAATCAAGCTGAAGTCA

  AAGGATCACTGCCTTGGATTCTACTGTGAATGATGACCTGGAAAATATCC

  TGAACAACAGCTTCAGGGTGATCATCAGAGACAAAAGTTCCAGAGCCAGg

  tagggaaaccctcaagccttgcaaagagcaaaatcatgccattgggttct

  taacctgctgagtgatttactatatgttactgtgggaggcaaagcgctca

  aatagcctgggtaagtatgtcaaataaaaagcaaaagtggtgtttcttga

  aatgttagacctgaggaaggaatattgataacttaccaataattttcaga

  atgatttatagatgtgcacttagtcagtgtctctccaccccgcacctgac

  aagcagtttagaatttattctaagaatctaggtttgctgggggctacatg

  ggaatcagcttcagtgaagagtttgttggaatgattcactaaattttcta

  tttccagcataaatccaagaacctctcagactagtttattgacactgctt

  ttcctccataatccatctcatctccgtccatcatggacactttgtagaat

  gacaggtcctggcagagactcacagatgcttctgaaacatcctttgcctt

  caaagaatgaacagcacacatactaaggatctcagtgatccacaaattag

  tttttgccacaatggttcttatgataaaagtctttcattaacagcaaatt

  gttttataatagttgttctgctttataataattgcatgcttcactttctt

  ttcttttctttttttttctttttttgctttttagtgccgcaggtgcagca

  tatgaaatttcccaggctaggggtcaaatcagaactacacctactggcct

  acgccacagccacagcaactcaggatctaagccatgtcggtgacctacac

  tacagctcatggcaatgccagatccttaacccaatgagcgaggccaggga

  tcgaacccatgtcctcatggatactagtcaggctcattatccgctgagcc

  ataacaggaactcccgagtttgctttttatcaaaattggtacagccttat

  tgtttctgaaaaccacaaaatgaatgtattcacataattttaaaaggtta

  aataatttatgatatacaagacaatagaaagagaaaacgtcattgcctct

  ttcttccacgacaacacgcctccttaattgatttgaagaaataactactg

  agcatggtttagtgtacttctttcagcaattagcctgtattcatagccat

  acatattcaattaaaatgagatcatgatatcacacaatacataccataca

  gcctatagggatttttacaatcatcttccacatgactacataaaaaccta

  cctaaaaaaaaaaaaaaccctacttcatcctcctattggctgctttgtgc

  tccattaaaaagctctatcataattaggttatgatgaggatttccatttt

  ctacctttcaagcaacatttcaatgcacagtcttatatacacatttgagc

  ctacttttctttttctttctttttttggtttttttttttttttttttttt

  ggtctttttgtcttttctaaggctgcatatggaggttcccaggctagctg

  tctaatcagaactatagctgctggcctacgccacatccacagcaatacaa

  gatctgagccatgtctgcaacttacaccacagctcacagcaacggtggat

  ccttaaaccactgagcaaggccagggatcaaacccataacttcatggctc

  ctagttggatttgttaaccactgagccatgatggcaactcctgagcctac

  ttttctaatcatttccaaccctaggacacttttttaagtttcatttttct

  ccccccaccccctgttttctgaagtgtgtttgcttccactgggtgacttc

  actcccaggatctcatctgcaggatactgcagctaagtgtatgagctctg

  aatttgaatcccaactctgccactcaaagggataggagtttccgatgtgg

  cccaatgggatcagtggcatctctgcagtgccaggacgcaggttccatcc

  ctggcccagcacagtgggttaagaatctggcattgctgcagctgaggcat

  agatttcaattgtgcctcagatctgatccttggcccaaggactgcatatg

  cctcagggcaaccaaaaaagagaaaaggggggtgatagcattagtttcta

  gatttgggggataattaaataaagtgatccatgtacaatgtatggcattt

  tgtaaatgctcaacaaatttcaactattatggagttcccatcatggctca

  gtggaagggaatctgattagcatccatgaggacacaggtccaaccccgac

  cttgctcagtgggcattgctgtgagctgtggcatgggttacagacgaagc

  tcggatctggcattgctgtggctgtggtgtaagccagcaactacagctct

  cattcagcccctagcctgggaacctccatatgcctaaaagacaaaaaata

  aaatttaaattaaaaataaagaaatgttaactattatgattggtactgct

  tgcattactgcaaagaaagtcactttctatactctttaatatcttagttg

  actgtgtgctcagtgaactattttggacacttaatttccactctcttcta

  tctccaacttgacaactctctttcctctcttctggtgagatccactgctg

  actttgctctttaaggcaactagaaaagtgctcagtgacaaaatcaaaga

  aagttaccttaatcttcagaattacaatcttaagttctcttgtaaagctt

  actatttcagtggttagtattattccttggtcccttacaacttatcagct

  ctgatctattgctgattttcaactatttattgttggagttttttcctttt

  ttccctgttcattctgcaaatgtttgctgagcatttgtcaagtgaagata

  ctggactgggccttccaaatataagacaatgaaacatcgggagttctcat

  tatggtgcagcagaaacgaatccaactaggaaatgtgaggttgcaggttc

  gatccctgcccttgctcagtgggttaaggatccagcattaccgtgagctg

  tggtgtaggttgcagacgtggctcagatcctgcgttgctgtggctgtggc

  ataggctggcagctctagctctgattcgaccgctagcctgggaacctcca

  tgcgccccgagtgcagcccttaaaaagcaaaaaaaaaagaaagaaagaaa

  aagacaatgaaacatcaaacagctaacaatccagtagggtagaaagaatc

  tggcaacagataagagcgattaaatgttctaggtccagtgaccttgcctc

  tgtgctctacacagtcgtgccacttgctgagggagaaggtctctcttgag

  ttgagtcctgaaagacattagttgttcacaaactaatgccagtgagtgaa

  ggtgtttccaagcagagggagagtttggtaaaaagctggaagtcacagaa

  agactctaaagagtttaggatggtgggagcaacatacgctgagatggggc

  tggaaggttaagagggaaacaactatagtaagtgaagctggactcacagc

  aaagtgaggacctcagcatccttgatggggttaccatggaaacaccaagg

  cacaccttgatttccaaaacagcaggcacctgattcagcccaatgtgaca

  tggtgggtacccctctagctctacctgttctgtgacaactgacaaccaac

  gaagttaagtctggattttctactctgctgatccttgtttttgtttcaca

  cgtcatctatagcttcatgccaaaatagagttcaaggtaagacgcgggcc

  ttggtttgatatacatgtagtctatcttgtttgagacaatatggtggcaa

  ggaagaggttcaaacaggaaaatactctctaattatgattaactgagaaa

  agctaaagagtcccataatgacactgaatgaagttcatcatttgcaaaag

  ccttcccccccccccaggagactataaaaaagtgcaattttttaaatgaa

  cttatttacaaaacagaaatagactcacagacataggaaacgaacagatg

  gttaccaagggtgaaagggagtaggagggataaataaggagtctggggtt

  agcagatacaccccagtgtacacaaaataaacaacagggacctactatat

  agcacagggaactatatgcagtagcttacaataacctataatggaaaaga

  atgtgaaaaagaatatatgtatgcgtgtgtgtgtaactgaatcactttgc

  tgtaacctgaatctaacataacattgtaaatcaactacagtttttttttt

  ttttaagtgcagggttttggtgttttttttttttcatttttgtttttgtt

  tttgttttttgctttttagggccacacccagacatatgggggttcccagg

  ctaggggtctaattagagctacagttgccggcttgcaccacagccacagc

  aacatcagatccgagccgcacttgcgacttacaccacagctcatggcaat

  accagatccttaacccactgagcaaggcccagggatcgtacccgcaacct

  catggttcctagtcagattcatttctgctgcgctacaatgggaactccaa

  gtgcagttttttgtaatgtgcttgtctttctttgtaattcatattcatcc

  tacttcccaataaataaataaatacataaataataaacataccattgtaa

  atcaactacaattttttttaaatgcagggtttttgttttttgttttttgt

  tttgtctttttgccttttctagggccgctcccatggcatatggaggttcc

  caggctaggggtcgaatcggagctgtagccaccggcctacgccagagcca

  cagcaacgcgggatccgagccgcgtctgcaacctacaccacagctcacgg

  caacgccggatcgttaacccactgagcaagggcagggatcgaacctgcaa

  cctcatggttcctagtcagattcgttaactactgagccacaacggaaact

  cctaaagtgcagtttttaaatgtgcttgtctttctttgtaatttacactc

  aacctacttcccaataaataaataaataaacaaataaatcatagacatgg

  ttgaattctaaaggaagggaccatcaggccttagacagaaatacgtcatc

  ttctagtattttaaaacacactaaagaagacaaacatgctctgccagaga

  agcccagggcctccacagctgcttgcaaagggagttaggcttcagtagct

  gacccaaggctctgttcctcttcagggaaaagggtttttgttcagtgaga

  cagcagacagctgtcactgtgGTGGACGTTCGGCCAAGGAACCAAGCTGG

  AACTCAAACgtaagtcaatccaaacgttccttccttggctgtctgtgtct

  tacggtctctgtggctctgaaatgattcatgtgctgactctctgaaacca

  gactgacattctccagggcaaaactaaagcctgtcatcaaactggaaaac

  tgagggcacattttctgggcagaactaagagtcaggcactgggtgaggaa

  aaacttgttagaatgatagtttcagaaacttactgggaagcaaagcccat

  gttctgaacagagctctgctcaagggtcaggaggggaaccagtttttgta

  caggagggaagttgagacgaacccctgtgTATATGGTTTCGGCGCGGGGA

  CCAAGCTGGAGCTCAAACgtaagtggctttttccgactgattctttgctg

  tttctaattgttggttggctttttgtccatttttcagtgttttcatcgaa

  ttagttgtcagggaccaaacaaattgccttcccagattaggtaccaggga

  ggggacattgctgcatgggagaccagagggtggctaatttttaacgtttc

  caagccaaaataactggggaagggggcttgctgtcctgtgagggtaggtt

  tttatagaagtggaagttaaggggaaatcgctatgGTTCACTTTTGGCTC

  GGGGACCAAAGTGGAGCCCAAAAttgagtacattttccatcaattatttg

  tgagatttttgtcctgttgtgtcatttgtgcaagtttttgacattttggt

  tgaatgagccattcccagggacccaaaaggatgagaccgaaaagtagaaa

  agagccaacttttaagctgagcagacagaccgaattgttgagtttgtgag

  gagagtagggtttgtagggagaaaggggaacagatcgctggctttttctc

  tgaattagcctttctcatgggactggcttcagagggggtttttgatgagg

  gaagtgttctagagccttaactgtgGGTTGTGTTCGGTAGCGGGACCAAG

  CTGGAAATCAAACgtaagtgcacttttctactcctttttctttcttatac

  gggtgtgaaattggggacttttcatgtttggagtatgagttgaggtcagt

  tctgaagagagtgggactcatccaaaaatctgaggagtaagggtcagaac

  agagttgtctcatggaagaacaaagacctagttagttgatgaggcagcta

  aatgagtcagttgacttgggatccaaatggccagacttcgtctgtaacca

  acaatctaatgagatgtagcagcaaaaagagatttccattgaggggaaag

  taaaattgttaatattgtgGATCACCTTTGGTGAAGGGACATCCGTGGAG

  ATTGAACgtaagtattttttctctactaccttctgaaatttgtctaaatg

  ccagtgttgacttttagaggcttaagtgtcagttttgtgaaaaatgggta

  aacaagagcatttcatatttattatcagtttcaaaagttaaactcagctc

  caaaaatgaatttgtagacaaaaagattaatttaagccaaattgaatgat

  tcaaaggaaaaaaaaattagtgtagatgaaaaaggaattcttacagctcc

  aaagagcaaaagcgaattaattttctttgaactttgccaaatcttgtaaa

  tgatttttgttctttacaatttaaaaaggttagagaaatgtatttcttag

  tctgttttctctcttctgtctgataaattattatatgagataaaaatgaa

  aattaataggatgtgctaaaaaatcagtaagaagttagaaaaatatatgt

  ttatgttaaagttgccacttaattgagaatcagaagcaatgttattttta

  aagtctaaaatgagagataaactgtcaatacttaaattctgcagagattc

  tatatcttgacagatatctcctttttcaaaaatccaatttctatggtaga

  ctaaatttgaaatgatcttcctcataatggagggaaaagatggactgacc

  ccaaaagctcagatttaaagaaatctgtttaagtgaaagaaaataaaaga

  actgcattttttaaaggcccatgaatttgtagaaaaataggaaatatttt

  aataagtgtattcttttattttcctgttattacttgatggtgtttttata

  ccgccaaggaggccgtggcaccgtcagtgtgatctgtagaccccatggcg

  gccttttttcgcgattgaatgaccttggcggtgggtccccagggctctgg

  tggcagcgcaccagccgctaaaagccgctaaaaactgccgctaaaggcca

  cagcaaccccgcgaccgcccgttcaactgtgctgacacagtgatacagat

  aatgtcgctaacagaggagaatagaaatatgacgggcacacgctaatgtg

  gggaaaagagggagaagcctgatttttattttttagagattctagagata

  aaattcccagtattatatccttttaataaaaaatttctattaggagatta

  taaagaatttaaagctatttttttaagtggggtgtaattctttcagtagt

  ctcttgtcaaatggatttaagtaatagaggcttaatccaaatgagagaaa

  tagacgcataaccctttcaaggcaaaagctacaagagcaaaaattgaaca

  cagcagccagccatctagccactcagattttgatcagttttactgagttt

  gaagtaaatatcatgaaggtataattgctgataaaaaaataagatacagg

  tgtgacacatctttaagtttcagaaatttaatggcttcagtaggattata

  tttcacgtatacaaagtatctaagcagataaaaatgccattaatggaaac

  ttaatagaaatatatttttaaattccttcattctgtgacagaaattttct

  aatctgggtcttttaatcacctaccctttgaaagagtttagtaatttgct

  atttgccatcgctgtttactccagctaatttcaaaagtgatacttgagaa

  agattatttttggtttgcaaccacctggcaggactattttagggccattt

  taaaactcttttcaaactaagtattttaaactgttctaaaccatttaggg

  ccttttaaaaatcttttcatgaatttcaaacttcgttaaaagttattaag

  gtgtctggcaagaacttccttatcaaatatgctaatagtttaatctgtta

  atgcaggatataaaattaaagtgatcaaggcttgacccaaacaggagtat

  cttcatagcatatttcccctcctttttttctagaattcatatgattttgc

  tgccaaggctattttatataatctctggaaaaaaaatagtaatgaaggtt

  aaaagagaagaaaatatcagaacattaagaattcggtattttactaactg

  cttggttaacatgaaggtttttattttattaaggtttctatctttataaa

  aatctgttcccttttctgctgatttctccaagcaaaagattcttgatttg

  ttttttaactcttactctcccacccaagggcctgaatgcccacaaagggg

  acttccaggaggccatctggcagctgctcaccgtcagaagtgaagccagc

  cagttcctcctgggcaggtggccaaaattacagttgacccctcctggtct

  ggctgaaccttgccccatatggtgacagccatctggccagggcccaggtc

  tccctctgaagcctttgggaggagagggagagtggctggcccgatcacag

  atgcggaaggggctgactcctcaaccggggtgcagactctgcagggtggg

  tctgggcccaacacacccaaagcacgcccaggaaggaaaggcagcttggt

  atcactgcccagagctaggagaggcaccgggaaaatgatctgtccaagac

  ccgttcttgcttctaaactccgagggggtcagatgaagtggttttgtttc

  ttggcctgaagcatcgtgttccctgcaagaagcggggaacacagaggaag

  gagagaaaagatgaactgaacaaagcatgcaaggcaaaaaaggccttagg

  atggctgcaggaagttagttcttctgcattggctccttactggctcgtcg

  atcgcccacaaacaacgcacccagtggagaacttccctgttacttaaaca

  ccattctctgtgcttgcttcctcagGGGCTGATGCCAAGCCATCCGTCTT

  CATCTTCCCGCCATCGAAGGAGCAGTTAGCGACCCCAACTGTCTCTGTGG

  TGTGCTTGATCAATAACTTCTTCCCCAGAGAAATCAGTGTCAAGTGGAAA

  GTGGATGGGGTGGTCCAAAGCAGTGGTCATCCGGATAGTGTCACAGAGCA

  GGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTCTCGCTGCCCA

  CGTCACAGTACCTAAGTCATAATTTATATTCCTGTGAGGTCACCCACAAG

  ACCCTGGCCTCCCCTCTGGTCACAAGCTTCAACAGGAACGAGTGTGAGGC

  TtagAGGCCCACAGGCCCCTGGCCTGCCCCCAGCCCCAGCCCCCCTCCCC

  ACCTCAAGCCTCAGGCCCTTGCCCCAGAGGATCCTTGGCAATCCCCCAGC

  CCCTCTTCCCTCCTCATCCCCTCCCCCTCTTTGGCTTTAACCGTGTTAAT

  ACTGGGGGGTGGGGGAATGAATAaataaaGTGAACCTTTGCACCTGTGAt

  ttctctctcctgtctgattttaaggttgttaaatgttgttttccccatta

  tagttaatcttttaaggaactacatactgagttgctaaaaactacaccat

  cacttataaaattcacgccttctcagttctcccctcccctcctgtcctcc

  gtaagacaggcctccgtgaaacccataagcacttctctttacaccctctc

  ctgggccggggtaggagactttttgatgtcccctcttcagcaagcctcag

  aaccattttgagggggacagttcttacagtcacat*tcctgtgatctaat

  gactttagttaccgaaaagccagtctctcaaaaagaagggaacggctaga

  aaccaagtcatagaaatatatatgtataaaatatatatatatccatatat

  gtaaaataacaaaataatgataacagcataggtcaacaggcaacagggaa

  tgttgaagtccattctggcacttcaatttaagggaataggatgccttcat

  tacattttaaatacaatacacatggagagcttcctatctgccaaagacca

  tcctgaatgccttccacactcactacaaggttaaaagcattcattacaat

  gttgatcgaggagttcccgttgtggctcagcaggttaagaacgtgactgg

  tatccaggaggatgcgggtttggtccccagcctcgctcagtggattaagg

  atccagtgttgctgcaagatcacgggctcagatcccgtgttctatggcta

  tggtgtaggctggtagctgcatgcagccctaatttgacccctagcctggg

  aactgccatatgccacatgtgaggcccttaaaacctaaaagaaaaaaaaa

  gaaaagaaatatcttacacccaatttatagataagagagaagctaaggtg

  gcaggcccaggatcaaagccctacctgcctatcttgacacctgatacaaa

  ttctgtcttctagggtttccaacactgcatagaacagagggtcaaacatg

  ctaccctcccagggactcctcccttcaaatgacataaattttgttgccca

  tctctgggggcaaaactcaacaatcaatggcatctctagtaccaagcaag

  gctcttctcatgaagcaaaactctgaagccagatccatcatgacccaagg

  aagtaaagacaggtgttactggttgaactgtatccttcaattcaatatgc

  tcaatttccaactcccagtccccgtaaatacaaccccctttgggaagaga

  gtccttgcagatgtagccacgttaaaaagagattatacagaaaggctagt

  gaggatgcagtgaaacgggatctttcatacattgctggtggaaatgtaaa

  atgctgcaggcactctagaaaataatttgccagttttttgaaaagctaaa

  caaaatagtttagttgcattctgggttatttatcccccagaaattaaaaa

  ttatgtccgcacaaaaacgtgtacataatcattcataacagccttgtac

  Seq ID No. 12

  caaggaaccaagctggaactcaaacgtaagtcaatccaaacgttccttcc

  ttggctgtctgtgtcttacggtctctgtggctctgaaatgattcatgtgc

  tgactctctgaaaccagactgacattctccagggcaaaactaaagcctgt

  catcaaactggaaaactgagggcacattttctgggcagaactaagagtca

  ggcactgggtgaggaaaaacttgttagaatgatagtttcagaaacttact

  gggaagcaaagcccatgttctgaacagagctctgctcaagggtcaggagg

  ggaaccagtttttgtacaggagggaagttgagacgaacccctgtgtatat

  ggtttcggcgcggggaccaagctggagctcaaacgtaagtggctttttcc

  gactgattctttgctgtttctaattgttggttggctttttgtccattttt

  cagtgttttcatcgaattagttgtcagggaccaaacaaattgccttccca

  gattaggtaccagggaggggacattgctgcatgggagaccagagggtggc

  taatttttaacgtttccaagccaaaataactggggaagggggcttgctgt

  cctgtgagggtaggtttttatagaagtggaagttaaggggaaatcgctat

  ggttcacttttggctcggggaccaaagtggagcccaaaattgagtacatt

  ttccatcaattatttgtgagatttttgtcctgttgtgtcatttgtgcaag

  tttttgacattttggttgaatgagccattcccagggacccaaaaggatga

  gaccgaaaagtagaaaagagccaacttttaagctgagcagacagaccgaa

  ttgttgagtttgtgaggagagtagggtttgtagggagaaaggggaacaga

  tcgctggctttttctctgaattagcctttctcatgggactggcttcagag

  ggggtttttgatgagggaagtgttctagagccttaactgtgggttgtgtt

  cggtagcgggaccaagctggaaatcaaacgtaagtgcacttttctactcc

  tttttctttcttatacgggtgtgaaattggggacttttcatgtttggagt

  atgagttgaggtcagttctgaagagagtgggactcatccaaaaatctgag

  gagtaagggtcagaacagagttgtctcatggaagaacaaagacctagtta

  gttgatgaggcagctaaatgagtcagttgacttgggatccaaatggccag

  acttcgtctgtaaccaacaatctaatgagatgtagcagcaaaaagagatt

  tccattgaggggaaagtaaaattgttaatattgtggatcacctttggtga

  agggacatccgtggagattgaacgtaagtattttttctctactaccttct

  gaaatttgtctaaatgccagtgttgacttttagaggcttaagtgtcagtt

  ttgtgaaaaatgggtaaacaagagcatttcatatttattatcagtttcaa

  aagttaaactcagctccaaaaatgaatttgtagacaaaaagattaattta

  agccaaattgaatgattcaaaggaaaaaaaaattagtgtagatgaaaaag

  gaattcttacagctccaaagagcaaaagcgaattaattttctttgaactt

  tgccaaatcttgtaaatgatttttgttctttacaatttaaaaaggttaga

  gaaatgtatttcttagtctgttttctctcttctgtctgataaattattat

  atgagataaaaatgaaaattaataggatgtgctaaaaaatcagtaagaag

  ttagaaaaatatatgtttatgttaaagttgccacttaattgagaatcaga

  agcaatgttatttttaaagtctaaaatgagagataaactgtcaatactta

  aattctgcagagattctatatcttgacagatatctcctttttcaaaaatc

  caatttctatggtagactaaatttgaaatgatcttcctcataatggaggg

  aaaagatggactgaccccaaaagctcagattt*aagaaaacctgtttaag

  *gaaagaaaataaaagaactgcattttttaaaggcccatgaatttgtaga

  aaaataggaaatattttaataagtgtattcttttattttcctgttattac

  ttgatggtgtttttataccgccaaggaggccgtggcaccgtcagtgtgat

  ctgtagaccccatggcggccttttttcgcgattgaatgaccttggcggtg

  ggtccccagggctctggtggcagcgcaccagccgctaaaagccgctaaaa

  actgccgctaaaggccacagcaaccccgcgaccgcccgttcaactgtgct

  gacacagtgatacagataatgtcgctaacagaggagaatagaaatatgac

  gggcacacgctaatgtggggaaaagagggagaagcctgatttttattttt

  tagagattctagagataaaattcccagtattatatccttttaataaaaaa

  tttctattaggagattataaagaatttaaagctatttttttaagtggggt

  gtaattctttcagtagtctcttgtcaaatggatttaagtaatagaggctt

  aatccaaatgagagaaatagacgcataaccctttcaaggcaaaagctaca

  agagcaaaaattgaacacagcagccagccatctagccactcagattttga

  tcagttttactgagtttgaagtaaatatcatgaaggtataattgctgata

  aaaaaataagatacaggtgtgacacatctttaagtttcagaaatttaatg

  gcttcagtaggattatatttcacgtatacaaagtatctaagcagataaaa

  atgccattaatggaaacttaatagaaatatatttttaaattccttcattc

  tgtgacagaaattttctaatctgggtcttttaatcacctaccctttgaaa

  gagtttagtaatttgctatttgccatcgctgtttactccagctaatttca

  aaagtgatacttgagaaagattatttttggtttgcaaccacctggcagga

  ctattttagggccattttaaaactcttttcaaactaagtattttaaactg

  ttctaaaccatttagggccttttaaaaatcttttcatgaatttcaaactt

  cgttaaaagttattaaggtgtctggcaagaacttccttatcaaatatgct

  aatagtttaatctgttaatgcaggatataaaattaaagtgatcaaggctt

  gacccaaacaggagtatcttcatagcatatttcccctcctttttttctag

  aattcatatgattttgctgccaaggctattttatataatctctggaaaaa

  aaatagtaatgaaggttaaaagagaagaaaatatcagaacattaagaatt

  cggtattttactaactgcttggttaacatgaaggtttttattttattaag

  gtttctatctttataaaaatctgttcccttttctgctgatttctccaagc

  aaaagattcttgatttgttttttaactcttactctcccacccaagggcct

  gaatgcccacaaaggggacttccaggaggccatctggcagctgctcaccg

  tcagaagtgaagccagccagttcctcctgggcaggtggccaaaattacag

  ttgacccctcctggtctggctgaaccttgccccatatggtgacagccatc

  tggccagggcccaggtctccctctgaagcctttgggaggagagggagagt

  ggctggcccgatcacagatgcggaaggggctgactcctcaaccggggtgc

  agactctgcagggtgggtctgggcccaacacacccaaagcacgcccagga

  aggaaaggcagcttggtatcactgcccagagctaggagaggcaccgggaa

  aatgatctgtccaagacccgttcttgcttctaaactccgagggggtcaga

  tgaagtggttttgtttcttggcctgaagcatcgtgttccctgcaagaagc

  ggggaacacagaggaaggagagaaaagatgaactgaacaaagcatgcaag

  gcaaaaaaggccttaggatggctgcaggaagttagttcttctgcattggc

  tccttactggctcgtcgatcgcccacaaacaacgcacccagtggagaact

  tccctgttacttaaacaccattctctgtgcttgcttcctcaggggctgat

  gccaagccatccgtcttcatcttcccgccatcgaaggagcagttagcgac

  cccaactgtctctgtggtgtgcttgatca

  Seq ID No. 15

  gatgccaagccatccgtcttcatcttcccgccatcgaaggagcagttagc

  gaccccaactgtctctgtggtgtgcttgatcaataacttcttccccagag

  aaatcagtgtcaagtggaaagtggatggggtggtccaaagcagtggtcat

  ccggatagtgtcacagagcaggacagcaaggacagcacctacagcctcag

  cagcaccctctcgctgcccacgtcacagtacctaagtcataatttatatt

  cctgtgaggtcacccacaagaccctggcctcccctctggtcacAAGCTTC

  AACAGGAACGAGTGTGAGGCTTAGAGGCCCACAGGCCCCTGGCCTGCCCC

  CAGCCCCAGCCCCCCTCCCCACCTCAAGCCTCAGGCCCTTGCCCCAGAGG

  ATCCTTGGCAATCCCCCAGCCCCTCTTCCCTCCTCATCCCCTCCCCCTCT

  TTGGCTTTAACCGTGTTAATACTGGGGGGTGGGGGAATGAATAAATAAAG

  TGAACCTTTGCACCTGTGATTTCTCTCTCCTGTCTGATTTTAAGGTTGTT

  AAATGTTGTTTTCCCCATTATAGTTAATCTTTTAAGGAACTACATACTGA

  GTTGCTAAAAACTACACCATCACTTATAAAATTCAcgCCTTCTCAGTTCT

  CCCCTCCCCTCCTGTCCTCCGTAAGACAGGCCTCCGTGAAACCCATAAGC

  ACTTCTCTTTACACCCTCTCCTGGGCCGGGGTAGGAGACTTTTTGATGTC

  CCCTcTTCAGCAAGCCTCAGAACCATTTTGAGGGGGACAGTTCTTACAGT

  CACAT*TCCtGtGATCTAATGACTTTAGTTaCCGAAAAGCCAGTCTCTCA

  AAAAGAAGGGAACGGCTAGAAACCAAGTCATAGAAATATATATGTATAAA

  ATATATATATATCCATATATGTAAAATAACAAAATAATGATAACAGCATA

  GGTCAACAGGCAACAGGGAATGTTGAAGTCCATTCTGGCACTTCAATTTA

  AGGGAATAGGATGCCTTCATTACATTTTAAATACAATACACATGGAGAGC

  TTCCTATCTGCCAAAGACCATCCTGAATGCTTTCCACACTCACTACAAGG

  TTAAAAGCATTCATTACAATGTTGATCGAGGAGTTCCCGTTGTGGCTCAG

  CAGGTTAAGAACGTGACTGGTATCCAGGAGGATGCGGGTTTGGTCCCCAG

  CCTCGCTCAGTGGATTAAGGATCCAGTGTTGCTGCAAGATCACGGGCTCA

  GATCCCGTGTTCTATGGCTATGGTGTAGGCTGGTAGCTGCATGCAGCCCT

  AATTTGACCCCTAGCCTGGGAACTGCCATAtGCCACATGTGAGGCCCTTA

  AAACCTAAAAGAAAAAaAAAGAAAAGAAATATCTTACACCCAATTTATAG

  ATAAGAGAGAAGCTAAGGTGGCAGGCCCAGGATCAAAGCCCTACCTGCCT

  ATCTTGACACCTGAtACAAATTCTGTCTTCTAGGGtTTCCAACACTGCAT

  AGAACAGAGGGTCAAACATGCTACCCTCCCAGGGACTCCTCCCTTCAAAT

  GACATAAATTTTGTTGCCCATCTCTGGGGGCAAAACTCAACAATCAATGG

  CATCTCTAGTACCAAGCAAGGCTCTTCTCATGAAGCAAAACTCTGAAGCC

  AGATCCATCATGACCCAAGGAAGTAAAGACAGGTGTTACTGGTTGAACTG

  TATCCTTCAATTCAATATGCTCAATTTCCAACTCCCAGTCCCCGTAAATA

  CAACCCCCTTTGGGAAGAGAGTCCTTGCAGATGTAGCCACGTTAAAAAGA

  GATTATACAGAAAGGCTAGTGAGGATGCAGTGAAACGGGATCTTTCATAC

  ATTGCTGGTGGAAATGTAAAATGCTGCAGGCACTCTAGAAAATAATTTGC

  CAGTTTTTTGAAAAGCTAAACAAAATAGTTTAGTTGCATTCTGGGTTATT

  TATCCCCCAGAAATTAAAAATTATGTCCGCACAAAAACGTGTACATAATC

  ATTCATAACAGCCTTGTACGAAAAGCTT

  Seq ID No. 16

  GGATCCTTAACCCACTAATCGAGGATCAAACACGCATCCTCATGGACAAT

  ATGTTGGGTTCTTAGCCTGCTGAGACACAACAGGAACTCCCCTGGCACCA

  CTTTAGAGGCCAGAGAAACAGCACAGATAAAATTCCCTGCCCTCATGAAG

  CTTATAGTCTAGCTGGGGAGATATCATAGGCAAGATAAACACATACAAAT

  ACATCATCTTAGGTAATAATATATACTAAGGAGAAAATTACAGGGGAGAA

  AGAGGACAGGAATTGCTAGGGTAGGATTATAAGTTCAGATAGTTCATCAG

  GAACACTGTTGCTGAGAAGATAACATTTAGGTAAAGACCGAAGTAGTAAG

  GAAATGGACCGTGTGCCTAAGTGGGTAAGACCATTCTAGGCAGCAGGAAC

  AGCGATGAAAGCACTGAGGTGGGTGTTCACTGCACAGAGTTGTTCACTGC

  ACAGAGTTGTGTGGGGAGGGGTAGGTCTTGCAGGCTCTTATGGTCACAGG

  AAGAATTGTTTTTACTCCCACCGAGATGAAGGTTGGTGGATTTGAGCAGA

  AGAATAATTCTGCCTGGTTTATATATAACAGGATTTCCCTGGGTGCTCTG

  ATGAGAATAATCTGTCAGGGGTGGGATAGGGAGAGATATGGCAATAGGAG

  CCTTGGCTAGGAGCCCACGACAATAATTCCAAGTGAGAGGTGGTGCTGCA

  TTGAAAGCAGGACTAACAAGACCTGCTGACAGTGTGGATGTAGAAAAAGA

  TAGAGGAGACGAAGGTGCATCTAGGGTTTTCTGCCTGAGGAATTAGAAAG

  ATAAAGCTAAAGCTTATAGAAGATGCAGCGCTCTGGGGAGAAAGACCAGC

  AGCTCAGTTTTGATCCATCTGGAATTAATTTTGGCATAAAGTATGAGGTA

  TGTGGGTTAACATTATTTGTTTTTTTTTTTTCCATGTAGCTATCCAACTG

  TCCCAGCATCATTTATTTTAAAAGACTTTCCTTTCCCCTATTGGATTGTT

  TTGGCACCTTCACTGAAGATCAACTGAGCATAAAATTGGGTCTATTTCTA

  AGCTCTTGATTCCATTCCATGACCTATTTGTTCATCTTTACCCCAGTAGA

  CACTGCCTTGATGATTAAAGCCCCTGTTACCATGTCTGTTTGGACATGGT

  AAATCTGAGATGCCTATTAGCCAACCAAGCAAGCACGGCCCTTAGAGAGC

  TAGATATGAGAGCCTGGAATTCAGACGAGAAAGGTCAGTCCTAGAGACAT

  ACATGTAGTGCCATCACCATGCGGATGGTGTTAAAAGCCATCAGACTGCA

  ACAGACTGTGAGAGGGTACCAAGCTAGAGAGCATGGATAGAGAAACCCAA

  GCACTGAGCTGGGAGGTGCTCCTACATTAAGAGATTAGTGAGATGAAGGA

  CTGAGAAGATTGATCAGAGAAGAAGGAaAATCAGGAAAATGGTGCTGTCc

  TGAAAATCCAAGGGAAGAGATGTTCCAAAGAGGAGAaAACTGATCAGTTG

  TCAGCTAGCGTCAATTGGGATGAAAATGGACCATTGGACAGAGGGATGTA

  GTGGGTCATGGGTGAATAGATAAGAGCAGCTTCTATAGAATGGCAGGGGC

  AAAATTCTCATCTGATCGGCATGGGTTcTAAAGAAAACGGGAAGAAAAAA

  TTGAGTGCATGACCAGTCCCTTCAAGTAGAGAGGTgGAAAAGGGAAGGAG

  GAAAATGAGGCCACGACAACATGAGAGAAATGACAGCATTTTTAAAAATT

  TTTTATTTTATTTTATTTATTTATTTTTGCTTTTTAGGGCTGCCCCTGCA

  Acatatggaggttcccaggttaggggtctaatcagagctatagctgccag

  cctacaccacagccatagcaatgccagatctacatgacctacaccacagc

  tcacagcaacgccggatccttaacccactgagtgaggccagagatcaaac

  ccatatccttatggatactagtcaggttcattaccactgagccaaaatgg

  gaaATCCTGAGTAATGACAGCATTTTTTAATGTGCCAGGAAGCAAAACTT

  GCCACCCCGAAATGTCTCTCAGGCATGTGGATTATTTTGAGCTGAAAACG

  ATTAAGGCCCAAAAAACACAAGAAGAAATGTGGACCTTCCCCCAACAGCC

  TAAAAAATTTAGATTGAGGGCCTGTTCCCAGAATAGAGCTATTGCCAGAC

  TTGTCTACAGAGGCTAAGGGCTAGGTGTGGTGGGGAAACCCTCAGAGATC

  AGAGGGACGTTTATGTACCAAGCATTGACATTTCCATCTCCATGCGAATG

  GCCTTCTTCCCCTCTGTAGCCCCAAACCACCACCCCCAAAATCTTCTTCT

  GTCTTTAGCTGAAGATGGTGTTGAAGGTGATAGTTTCAGCCACTTTGGCG

  AGTTCCTCAGTTGTTCTGGGTCTTTCCTCCGGATCCACATTATTCGACTG

  TGTTTGATTTTTTCCTGTTTATCTGTCTCATTGGCACCCATTTCATTCTT

  AGACCAGCCCAAAGAACCTAGAAGAGTGAAGGAAAATTTCTTCCACCCTG

  ACAAATGCTAAATGAGAATCACCgCAGTAGAGGAAAATGATCTGGTgCTG

  CGGGAGATAGAAGAGAAAATcGCTGGAGAGATGTCACTGAGTAGGTGAGA

  TGGGAAAGGGGGGGCACAGGTGGAGGTGTTGCCCTCAGCTAGGAAGACAG

  ACAGTTcacagaagagaagcgggtgtccgtGGACATCTTGCCTCATGGAT

  GAGGAAACCGAGGCTAAGAAAGACTGCAAAAGAAAGGTAAGGATTGCAGA

  GAGGTCGATCCATGACTAAAATCACAGTAACCAACCCCAAACCACCATGT

  TTTCTCCTAGTCTGGCACGTGGCAGGTACTGTGTAGGTTTTCAATATTAT

  TGGTTTGTAACAGTACCTATTAGGCCTCCATCcCCTCCTCTAATACTAAC

  AAAAGTGTGAGACTGGTCAGTGAAAAATGGTCTTCTTTCTCTATGCAATC

  TTTCTCAAGAAGATACATAACTTTTTATTTTATCATaGGCTTGAAGAGCA

  AATGAGAAACAgCCTCCAACCTATGACACCGTAACAAAGTGTTTATGATC

  AGTGAAGGGCAAGAAACAAAACATACACaGTAAAGACCCTCCATAATATT

  GtGGGCTGGCCCAaCACAGGCCAGGTTGTAAAAGCTTTTTATTCTTTGAT

  AGAGGAATGGATAGTAATGTTTCAACCTGGACAGAGAT*CATGTTCACTG

  AATCCTTCCAAAAATTCATGGGTAGTTTGAAtTATAAGGAAAATAAGACT

  TAGGATAAATACTTTgTCCA*GATCCCAGAGTTAATgCCAAAATCAGTTT

  TCAGACTCCAGGCAGCCTGATCAAGAGCCTAAACTTTAAAGACACAGTCC

  CTTAATAACTACTATTCACAGTTGCACTTTCAgGGCGCAAAGACTCATTG

  AATCCTACAATAGAATGAGTTTAGATATCAAATCTCTCAGTAATAGATGA

  GGAGACTAAATAGCGGGCATGACCTGGTCACTTAAAGACAGAATTGAGAT

  TCAAGGCTAGTGTTCTTTCTACCTGTTTTGTTTCTACAAGATGTAGCAAT

  GCGCTAATTACAGACCTCTCAGGGAAGGAATTCACAACCCTCAGCAAAAA

  CCAAAGACAAATCTAAGACAACTAAGAGTGTTGGTTTAATTTGGAAAAAT

  AACTCACTAACCAAACGCCCCTCTTAGCACCCCAATGTCTTCCACCATCA

  CAGTGCTCAGGCCTCAACCATGCCCCAATCACCCCAGCCCCAGACTGGTT

  ATTACCAAGTTTCATGATGACTGGCCTGAGAAGATCAAAAAAGCAATGAC

  ATCTTACAGGGGACTACCCCGAGGACCAAGATAGCAACTGTCATAGCAAC

  CGTCACACTGCTTTGGTCA

  Seq ID No. 19

  ggatcaaacacgcatcctcatggacaatatgttgggttcttagcctgctg

  agacacaacaggaactcccctggcaccactttagaggccagagaaacagc

  acagataaaattccctgccctcatgaagcttatagtctagctggggagat

  atcataggcaagataaacacatacaaatacatcatcttaggtaataatat

  atactaaggagaaaattacaggggagaaagaggacaggaattgctagggt

  aggattataagttcagatagttcatcaggaacactgttgctgagaagata

  acatttaggtaaagaccgaagtagtaaggaaatggaccgtgtgcctaagt

  gggtaagaccattctaggcagcaggaacagcgatgaaagcactgaggtgg

  gtgttcactgcacagagttgttcactgcacagagttgtgtggggaggggt

  aggtcttgcaggctcttatggtcacaggaagaattgttttactcccaccg

  agatgaaggttggtggattttgagcagaagaataattctgcctggtttat

  atataacaggatttccctgggtgctctgatgagaataatctgtcaggggt

  gggatagggagagatatggcaataggagccttggctaggagcccacgaca

  ataattccaagtgagaggtggtgctgcattgaaagcaggactaacaagac

  ctgctgacagtgtggatgtagaaaaagatagaggagacgaaggtgcatct

  agggttttctgcctgaggaattagaaagataaagctaaagcttatagaag

  atgcagcgctctggggagaaagaccagcagctcagttttgatccatctgg

  aattaattttggcataaagtatgaggtatgtgggttaacattatttgttt

  tttttttttccatgtagctatccaactgtcccagcatcatttattttaaa

  agactttcctttcccctattggattgttttggcaccttcactgaagatca

  actgagcataaaattgggtctatttctaagctcttgattccattccatga

  cctatttgttcatctttaccccagtagacactgccttgatgattaaagcc

  cctgttaccatgtctgttttggacatggtaaatctgagatgcctattagc

  caaccaagcaagcacggcccttagagagctagatatgagagcctggaatt

  cagacgagaaaggtcagtcctagagacatacatgtagtgccatcaccatg

  cggatggtgttaaaagccatcagactgcaacagactgtgagagggtacca

  agctagagagcatggatagagaaacccaagcactgagctgggaggtgctc

  ctacattaagagattagtgagatgaaggactgagaagattgatcagagaa

  gaaggaaaatcaggaaaatggtgctgtcctgaaaatccaagggaagagat

  gttccaaagaggagaaaactgatcagttgtcagctagcgtcaattgggat

  gaaaatggaccattggacagagggatgtagtgggtcatgggtgaatagat

  aagagcagcttctatagaatggcaggggcaaaattctcatctgatcggca

  tgggttctaaagaaaacgggaagaaaaaattgagtgcatgaccagtccct

  tcaagtagagaggtggaaaagggaaggaggaaaatgaggccacgacaaca

  tgagagaaatgacagcatttttaaaaattttttattttattttatttatt

  tatttttgctttttagggctgcccctgcaacatatggaggttcccaggtt

  aggggtctaatcagagctatagctgccagcctacaccacagccatagcaa

  tgccagatctacatgacctacaccacagctcacagcaacgccggatcctt

  aacccactgagtgaggccagagatcaaacccatatccttatggatactag

  tcaggttcattaccactgagccaaaatgggaaatcctgagtaatgacagc

  attttttaatgtgccaggaagcaaaacttgccaccccgaaatgtctctca

  ggcatgtggattattttgagctgaaaacgattaaggcccaaaaaacacaa

  gaagaaatgtggaccttcccccaacagcctaaaaaatttagattgagggc

  ctgttcccagaatagagctattgccagacttgtctacagaggctaagggc

  taggtgtggtggggaaaccctcagagatcagagggacgtttatgtaccaa

  gcattgacatttccatctccatgcgaatggccttcttcccctctgtagcc

  ccaaaccaccacccccaaaatcttcttctgtctttagctgaagatggtgt

  tgaaggtgatagtttcagccactttggcgagttcctcagttgttctgggt

  ctttcctccTgatccacattattcgactgtgtttgattttctcctgttta

  tctgtctcattggcacccatttcattcttagaccagcccaaagaacctag

  aagagtgaaggaaaatttcttccaccctgacaaatgctaaatgagaatca

  ccgcagtagaggaaaatgatctggtgctgcgggagatagaagagaaaatc

  gctggagagatgtcactgagtaggtgagatgggaaaggggtgacacaggt

  ggaggtgttgccctcagctaggaagacagacagttcacagaagagaagcg

  ggtgtccgtggacatcttgcctcatggatgaggaaaccgaggctaagaaa

  gactgcaaaagaaaggtaaggattgcagagaggtcgatccatgactaaaa

  tcacagtaaccaaccccaaaccaccatgttttctcctagtctggcacgtg

  gcaggtactgtgtaggttttcaatattattggtttgtaacagtacctatt

  aggcctccatcccctcctctaatactaacaaaagtgtgagactggtcagt

  gaaaaatggtcttctttctctatgaatctttctcaagaagatacataact

  ttttattttatcataggcttgaagagcaaatgagaaacagcctccaacct

  atgacaccgtaacaaaatgtttatgatcagtgaagggcaagaaacaaaac

  atacacagtaaagaccctccataatattgtgggtggcccaacacaggcca

  ggttgtaaaagctttttattctttgatagaggaatggatagtaatgtttc

  aacctggacagagatcatgttcactgaatccttccaaaaattcatgggta

  gtttgaattataaggaaaataagacttaggataaatactttgtccaagat

  cccagagttaatgccaaaatcagttttcagactccaggcagcctgatcaa

  gagcctaaactttaaagacacagtcccttaataactactattcacagttg

  cactttcagggcgcaaagactcattgaatcctacaatagaatgagtttag

  atatcaaatctctcagtaatagatgaggagactaaatagcgggcatgacc

  tggtcacttaaagacagaattgagattcaaggctagtgttctttctacct

  gttttgtttctacaagatgtagcaatgcgctaattacagacctctcaggg

  aaggaattcacaaccctcagcaaaaaccaaagacaaatctaagacaacta

  agagtgttggtttaatttggaaaaataactcactaaccaaacgcccctct

  tagcaccccaatgtcttccaccatcacagtgctcaggcctcaaccatgcc

  ccaatcacc

  Seq ID No. 25

  GCACATGGTAGGCAAAGGACTTTGCTTCTCCCAGCACATCTTTCTGCAGA

  GATCCATGGAAACAAGACTCAACTCCAAAGCAGCAAAGAAGCAGCAAGTT

  CTCAAGTGATCTCCTCTGACTCCCTCCTCCCAGGCTAATGAAGCCATGTT

  GCCCCTGGGGGATTAAGGGCAGGTGTCCATTGTGGCACCCAGCCCGAAGA

  CAAGCAATTTGATCAGGTTCTGAGCACTCCTGAATGTGGACTCTGGAATT

  TTCTCCTCACCTTGTGGCATATCAGCTTAAGTCAAGTACAAGTGACAAAC

  AACATAATCCTAAGAAGAGAGGAATCAAGCTGAAGTCAAAGGATCACTGC

  CTTGGATTCTACTGTGAATGATGACCTGGAAAATATCCTGAACAACAGCT

  TCAGGGTGATCATCAGAGACAAAAGTTCCAGAGCCAGGTAGGGAAACCCT

  CAAGCCTTGCAAAGAGCAAAATCATGCCATTGGGTTCTTAACCTGCTGAG

  TGATTTACTATATGTTACTGTGGGAGGCAAAGCGCTCAAATAGCCTGGGT

  AAGTATGTCAAATAAAAAGCAAAAGTGGTGTTTCTTGAAATGTTAGACCT

  GAGGAAGGAATATTGATAACTTACCAATAATTTTCAGAATGATTTATAGA

  TGTGCACTTAGTCAGTGTCTCTCCACCCCGCACCTGACAAGCAGTTTAGA

  ATTTATTCTAAGAATCTAGGTTTGCTGGGGGCTACATGGGAATCAGCTTC

  AGTGAAGAGTTTGTTGGAATGATTCACTAAATTTTCTATTTCCAGCATAA

  ATCCAAGAACCTCTCAGACTAGTTTATTGACACTGCTTTTCCTCCATAAT

  CCATCTCATCTCCGTCCATCATGGACACTTTGTAGAATGACAGGTCCTGG

  CAgAGACTCaCAGATGCTTCTGAAACATCCTTTGCCTTCAAAGAATGAAC

  AGCACACATACTAAGGATCTCAGTGATCCACAAATTAGTTTTTGCCACAA

  TGGTTCTTATGATAAAAGTCTTTCATTAACAGCAAATTGTTTTATAATAG

  TTGTTCTGCTTTATAATAATTGCATGCTTCACTTTCTTTTCTTTTCTTTT

  TTTTTCTTTTTTTGCTTTTTAGTGCCGCAGGTgcagcatatgaaatttcc

  caggctaggggtcaaatcagaactacacctactggcctacgccacagcca

  cagcaactcaggatctaagccatgtcggtgacctacactacagctcatgg

  caatgccagatccttaacccaatgagcgaggccagggatcgaacccatgt

  cctcatggatactagtcaggctcattatccgctgagccataacaggaact

  cccGAGTTTGCTTTTTATCAAAATTGGTACAGCCTTATTGTTTCTGAAAA

  CCACAAAATGAATGTATTCACATAATTTTAAAAGGTTAAATAATTTATGA

  TATACAAGACAATAGAAAGAGAAAACGTCATTGCCTCTTTCTTCCACGAC

  AACACGCCTCCTTAATTGATTTGAAGAAATAACTACTGAGCATGGTTTAG

  TGTACTTCTTTCAGCAATTAGCCTGTATTCATAGCCATACATATTCAATT

  AAAATGAGATCATGATATCACACAATACATACCATACAGCCTATAGGGAT

  TTTTACAATCATCTTCCACATGACTACATAAAAACCTACCTAAAAAAAAA

  AAAAACCCTACTTCATCCTCCTATTGGCTGCTTTGTGCTCCATTAAAAAG

  CTCTATCATAATTAGGTTATGATGAGGATTTCCATTTTCTACCTTTCAAG

  CAACATTTCAATGCACAGTCTTATATACACATTTGAGCCTACTTTTCTTT

  TTCTTTCTTTTTTTGGTTTTTTTTTTTTTTTTTTTTTTGGTCTTTTTGTC

  TTTTCTAAGgctgcatatggaggttcccaggctagctgtctaatcagaac

  tatagctgctggcctacgccacatccacagcaatacaagatctgagccat

  gtctgcaacttacaccacagctcacagcaacggtggatccttaaaccact

  gagcaaggccagggatcaaacccatAACTTCATGGCTCCTAGTTGGATTT

  GTTAACCACTGAGCCATGATGGCAACTCCTGAGCCTACTTTTCTAATCAT

  TTCCAACCCTAGGACACTTTTTTAAGTTTCATTTTTCTCCCCCCACCCCC

  TGTTTTCTGAAGTGTGTTTGCTTCCACTGGGTGACTTCACTCCCAGGATC

  TCATCTGCAGGATACTGCAGCTAAGTGTATGAGCTCTGAATTTGAATCCC

  AACTCTGCCACTCAAAGGGATAGGAGTTTCCGATGTGGCCCAATGGGATC

  AGTGGCATCTCTGCAGTGCCAGGACGCaggttccatccctggcccagcac

  agtgggttaagaatctggCATTGCTGCAGCTGAGGCATAGATTTCAATTG

  TGCCTCAgATCTGATCCTTGGCCCAAGGACTGCATATGCCTCAGGGCAAC

  CAAAAAAGAGAAAAGGGGGGTGATAGCATTAGTTTCTAGATTTGGGGGAT

  AATTAAATAAAGTGATCCATGTACAATGTATGGCATTTTGTAAATGCTCA

  ACAAATTTCAACTATTATggagttcccatcatggctcagtggaagggaat

  ctgattagcatccatgaggacacaggtCCAACCCCGACCTTGCTCAGTGG

  GCATTGCTGTGAGCTGTGGCATGGGTTACAGACGAAGCTCGGATCTGGCA

  TTGCTGTGGCTGTGGTGTAAGCCAgCAActacagctctcattcagcccct

  agcctgggaacctccatatgccTAAAAGACAAAAAATAAAATTTAAATTA

  AAAATAAAGAAATGTTAACTATTATGATTGgTACTGCTTGCATTACTGCA

  AAGAAAGTCACTTTCTATACTCTTTAATATCTTAGTTGACTGTGTGCTCA

  GTGAACTATTTTGGACACTTAATTTCCACTCTCTTCTATCTCCAACTTGA

  CAACTCTCTTTCCTCTCTTCTGGTGAGATCCACTGCTGACTTTGCTCTTT

  AAGGCAACTAGAAAAGTGCTCAGTGACAAAATCAAAGAAAGTTACCTTAA

  TCTTCAGAATTACAATCTTAAGTTCTCTTGTAAAGCTTACTATTTCAGTG

  GTTAGTATTATTCCTTGGTCCCTTACAACTTATCAGCTCTGATCTATTGC

  TGATTTTCAACTATTTATTGTTGGAGTTTTTCCTTTTTTTCCCTGTTCAT

  TCTGCAAATGTTTGCTGAGCATTTGTCAAGTGAAGATACTGGACTGGGCC

  TTCCAAATATAAGACAATGAAACATCGGGAGTTCTCATTATGGTGCAGCA

  GAaacgaatccaactaggaaatgtgaggttgcaggttcgatccctgccct

  tgctcagtgggttaaggatccagcattaccgtgagctgtggtgtaggttg

  cagacgtggctcagatcctgcgttgctgtggctgtggcataggctggcag

  ctctagctctgattcgaccgctagcctgggaacctccatGCGCCCCGAGT

  GCAGCCCTTAAAAAGCAAAAAAAAAAGAAAGAAAGAAAAAGACAATGAAA

  CATCAAACAGCTAACAATCCAGTAGGGTAGAAAGAATCTGGCAACAGATA

  AGAGCGATTAAATGTTCTAGGTCCAGTGACCTTGCCTCTGTGCTCTACAC

  AGTCGTGCCACTTGCTGAGGGAGAAGGTCTCTCTTGAGTTGAGTCCTGAA

  AGACATTAGTTGTTCACAAACTAATGCCAGTGAGTGAAGGTGTTTCCAAG

  CAGAGGGAGAGTTTGGTAAAAAGCTGGAAGTCACAGAAAGACTCTAAAGA

  GTTTAGGATGGTGGGAGCAACATACGCTGAGATGGGGCTGGAAGGTTAAG

  AGGGAAACAACTATAGTAAGTGAAGCTGGACTCACAGCAAAGTGAGGACC

  TCAGCATCCTTGATGGGGTTACCATGGAAACACCAAGGCACACCTTGATT

  TCCAAAACAGCAGGCACCTGATTCAGCCCAATGTGACATGGTGGGTACCC

  CTCTAGCTCTACCTGTTCTGTGACAACTGACAACCAACGAAGTTAAGTCT

  GGATTTTCTACTCTGCTGATCCTTGTTTTTGTTTCACACGTCATCTATAG

  CTTCATGCCAAAATAGAGTTCAAGGTAAGACGCGGGCCTTGGTTTGATAT

  ACATGTAGTCTATCTTGTTTGAGACAATATGGTGGCAAGGAAGAGGTTCA

  AACAGGAAAATACTCTCTAATTATGATTAACTGAGAAAAGCTAAAGAGTC

  CCATAATGACACTGAATGAAGTTCATCATTTGCAAAAGCCTTCCCCCCCC

  CCCAGGAGACTATAAAAAAGTGCAATTTTTTAAATGAACTTATTTACAAA

  ACAGAAATAGACTCACAGACATAGGAAACGAACAGATGGTTACCAAGGGT

  GAAAGGGAGTAGGAGGGATAAATAAGGAGTCTGGGGTTAGCAGATACACC

  CCAGTGTACACAAAATAAACAACAGGGACCTACTATATAGCACAGGGAAC

  TATATGCAGTAGCTTACAATAACCTATAATGGAAAAGAATGTGAAAAAGA

  ATATATGTATGCGTGTGTGTGTAACTGAATCACTTTGCTGTAACCTGAAT

  CTAACATAACATTGTAAATCAACTACAGTTTTTTTTTTTTTTAAGTGCAG

  GGTTTTGGTGTTTTTTTTTTTTCATTTTTGTTTTTGTTTTTGTTTTTTGC

  TTTTTAGGGCCACACCCAGACATATGGGGGTTCCCAGGctAGGGGTcTAa

  TTAGAGcTACAGtTGCCGGCTTGCAccacagccacagcaacatcagatcc

  gagccgcacttgcgacttacaccacagctcatggcaataccagatcctta

  acccactgagcaaggcccagggatcgtacccgcaacctcatggttcctag

  tcagattcattTCTGCTGCGCTACAATGGGAACTCCAAGTGCAGTTTTTT

  GTAATGTGCTTGTCTTTCTTTGTAATTCATATTCATCCTACTTCCCAATA

  AATAAATAAATACATAAATAATAAACATACCATTGTAAATCAACTACAAT

  TTTTTTTAAATGCAGGGTTTTTGTTTTTTGTTTTTTGTTTTGTCTTTTTG

  CCTTTTCTAgggccgctcccatggcatatggaggttcccaggctaggggt

  cgaatcggagctgtagccaccggcctacgccagagccacagcaacgcggg

  atccgagccgcgtctgcaacctacaccacagctcacggcaacgccggatc

  gttaacccactgagcaagggcagggatcgaacctgcaacctcatggttcc

  tagtcagattcgttaactactgagccacaacggaaacTCCTAAAGTGCAG

  TTTTTAAATGTGCTTGTCTTTCTTTGTAATTTACACTCAACCTACTTCCC

  AATAAATAAATAAATAAACAAATAAATCATAGACATGGTTGAATTCTAAA

  GGAAGGGACCATCAGGCCTTAGACAGAAATACGTCATCTTCTAGTATTTT

  AAAACACACTAAAGAAGACAAACATGCTCTGCCAGAGAAGCCCAGGGCCT

  CCACAGCTGCTTGCAAAGGGAGTTAGGCTTCAGTAGCTGACCCAAGGCTC

  TGTTCCTCTTCAGGGAAAAGGGTTTTTGTTCAGTGAGACAGCAGACAGCT

  GTCACTGTGgtggacgttcggccaaggaaccaagctggaactcaaacGTA

  AGTCAATCCAAACGTTCCTTCCTTGGCTGTCTGTGTCTTACGGTCTCTGT

  GGCTCTGAAATGATTCATGTGCTGACTCTCTGAAACCAGACTGACATTCT

  CCAGGGCAAAACTAAAGCCTGTCATCAAACcGGAAAACTGAGGGCACATT

  TTCTGGGCAGAACTAAGAGTCAGGCACTGGGTGAGGAAAAACTTGTTAGA

  ATGATAGTTTCAGAAACTTACTGGGAAGCAAAGCCCATGTTCTGAACAGA

  GCTCTGCTCAAGGGTCAGGAGGGGAACCAGTTTTTGTACAGGAGGGAAGT

  TGAGACGAACCCCTGTGTAtatggtttcggcgcggggaccaagctggagc

  tcaaacGTAAGTGGCTTTTTCCGACTGATTCTTTGCTGTTTCTAATTGTT

  GGTTGGCTTTTTGTCCATTTTTCAGTGTTTTCATCGAATTAGTTGTCAGG

  GACCAAACAAATTGCCTTCCCAGATTAGGTACCAGGGAGGGGACATTGCT

  GCATGGGAGACCAGAGGGTGGCTAATTTTTAACGTTTCCAAGCCAAAATA

  ACTGGGGAAGGGGGCTTGCTGTCCTGTGAGGGTAGGTTTTTATAGAAGTG

  GAAGTTAAGGGGAAATCGCTATGGTtcacttttggctcggggaccaaagt

  ggagcccaaaattgaGTACATTTTCCATCAATTATTTGTGAGATTTTTGT

  CCTGTTGTGTCATTTGTGCAAGTTTTTGACATTTTGGTTGAATGAGCCAT

  TCCCAGGGACCCAAAAGGATGAGACCGAAAAGTAGAAAAGAGCCAACTTT

  TAAGCTGAGCAGACAGACCGAATTGTTGAGTTTGTGAGGAGAGTAGGGTT

  TGTAGGGAGAAAGGGGAACAGATCGCTGGCTTTTTCTCTGAATTAGCCTT

  TCTCATGGGACTGGCTTCAGAGGGGGTTTTTGATGAGGGAAGTGTTCTAG

  AGCCTTAACTGTGGgttgtgttcggtagcgggaccaagctggaaatcaaa

  CGTAAGTGCACTTTTCTACTCC

Porcine Lambda Light Chain
• In another embodiment, novel genomic sequences encoding the lambda light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate lambda light chain regions. In one embodiment, the porcine lambda light chain nucleotides include a concatamer of J to C units. In a specific embodiment, an isolated porcine lambda nucleotide sequence is provided, such as that depicted in Seq ID No. 28. See FIG. 3 for a diagram of the organization of the porcine lamba immunoglobulin locus.
• In one embodiment, nucleotide sequence is provided that includes 5′ flanking sequence to the first lambda J/C region of the porcine lambda light chain genomic sequence, for example, as represented by Seq ID No 32.
• Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, approximately 200 base pairs downstream of lambda J/C, such as that represented by Seq ID No 33. Alternatively, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, approximately 11.8 kb downstream of the J/C cluster, near the enhancer (such as that represented by Seq ID No. 34), approximately 12 Kb downstream of lambda, including the enhancer region (such as that represented by Seq ID No. 35), approximately 17.6 Kb downstream of lambda (such as that represented by Seq ID No. 36, approximately 19.1 Kb downstream of lambda (such as that represented by Seq ID No. 37), approximately 21.3 Kb downstream of lambda (such as that represented by Seq ID No. 38), and/or approximately 27 Kb downstream of lambda (such as that represented by Seq ID No. 39).
• In still further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25, 30, 40, 50, 75, 100, 150, 200, 250, 500 or 1,000 contiguous nucleotides of Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39, as well as, nucleotides homologous thereto.

• Seq ID No. 28

  CCTTCCTCCTGCACCTGTCAACTCCCAATAAACCGTCCTCCTTGTCATTC

  AGAAATCATGCTCTCCGCTCACTTGTGTCTACCCATTTTCGGGCTTGCAT

  GGGGTCATCCTCGAAGGTGGAGAGAGTCCCCCTTGGCCTTGGGGAAGTCG

  AGGGGGGCGGGGGGAGGCCTGAGGCATGTGCCAGCGAGGGGGGTCACCTC

  CACGCCCCTGAGGACCTTCTAGAACCAGGGGCGTGGGGCCACCGCCTGAG

  TGGAAGGCTGTCCACTTTTCCCCCGGGCCCCCAGGCTCCCTCCTCCGTGT

  GGACCTTGTCCACCTCTGACTGGCCCAGCCACTCATGCATTGTTTCCCCG

  AAACCCCAGGACGATAGCTCAGCACGCGACAGTGTCCCCCTCTGAGGGCC

  TCTGTCCATTTCAGGACGACCCGCATGTACAGCGTGACCACTCTGCTCAC

  GCCCACTCACCACGTCCTAGAGCCCCACCCCCAGCCCCATCCTTAGGGGC

  ACAGCCAGcTCCGACCGCCCCGGGGACACCACCCTCTGCCCCTTcCCCAG

  GCCCTCCCTGTCACACGCACCACAGGGCCCTCCGTCCCGAGACCCTGCTC

  CCTCATCCCTCGGTCCCCTCAGGTAGCCTTCCACCCGCGTGTGTCCCGAG

  GTCCCAGATGCAGCAAGGCCCCTGGGACAACGCCAGATCTCTGCTCTcCC

  CGACCCCTCAGAAGCCAGCCCACGCCTGGCCCCACCACCACTGCCTAACg

  TCCAAGTGTCCATAGGCCTCGGGACCTCCAAGTCCAGGTTCTGCCTCTGG

  GATTCCGCCATGGGTCTGCCTGGGAAATGATGCACTTGGAGGAGCTCAGC

  ATGGGATGCGGGACCTTGTCTCTAGGCGCTcCCTCAGGATCCCACAGCTG

  CCCTGTGAGACACACACACACACACACACACACACACACACACACACACA

  CACACAAACACGCATGCACGCACGCCGGCACACACGCTATTGCAGAGATG

  GCCACGGTAGCTGTGCCTCGAGGCCGAGTGGAGTGTCTAGAACTCTCGGG

  GGTCCCCTCTGCAGACGACACTGCTCCATCCCCCCCGTGCCCTGAAGGGC

  TCCTCACTCTCCCATCAGGATCTCTCCAAGCTGCTGACCTGGAGAGGAAG

  GGGCCTGGGACAGGCGGGGACACTCAGACCTCCCTGCTGCCCCTCCTCTG

  CCTGGGCTTGGACGGCTCCCCCCTTCCCACGGGTGAAGGTGCAGGTGGGG

  AGAGGGCACCCCCCTCAGCCTCCCAGACCCAGACCAGCCCCCGTGGCAGG

  GGCAGCCTGTGAGCCTCCAGCCAGATGCAGGTGGCCTGGGGTGGGGGGTG

  GAGGGGGCGGGAGGTTTATGTTTGAGGCTGTATCACTGTGTAATATTTTC

  GGCGGTGGGACCCATCTGACCGTCCTCGGTGAGTCTCCCCTTTTCTCTCC

  TCCTTGGGGATCCGAGTGAAATCTGGGTCGATCTTCTCTCCGTTCTCCTC

  CGACTGGGGCTGAGGTCTGAACCTCGGTGGGGTCCGAAGAGGAGGCCCCT

  AGGCCAGGCTCCTCAGCCCCTCCAGCCCGACcgGCCCTCTTGACACAGGG

  TCCAGCTAAGGGCAGACATGGAGGCTGCTAGTCCAGGGCCAGGCTCTGAG

  ACCCAAGGGCGCTGCCCAAGGAACCCTTGCCCCAGGGACCCTGGGAGCAA

  AGCTCCTCACTCAGAGCCTGCAGCCCTGGGGTCTGAGGACAAGGAGGGAC

  TGAGGACTGGGCGTGGGGAGTTCAGGCGGGGACACCAGGTCCAGGGAGGT

  GACAAAGGCGCTGGGAGGGGGCGGACGGTGCCGGGGACTCCTCCTGGGCC

  CTGTGGGCTCGGGGTCCTTGTGAGGACCCTGAGGGACTGAGGGGCCCCTG

  GGCCTAGGGACTTGCAgTgAGGGAGGCAGGGAGTGTCCCTTGAGAACGTG

  GCCTCCGCGGGCTGGGTCCCCCTCGTGCTCCCAGCC*GGGAGGACACCCC

  AGAGCAAGCGCCCCAGGTGGGCGGGGAGGGTCTCCTCACAGGGGCAGCTG

  ACAGATAGAGGCCCCCGCCAGGCAGATGCTTGATCCTGGCAgTTATACTG

  GGTTC**GCACAACTTTCCCTGAACAAGGGGCCCTCCGAACAGACACAGA

  CGCAACCCAGTCGACCcaggCTCAGCACAgAAAATGCACTGACACCCAAA

  ACCCTCATCTggggGCCTGGCCGGcAtCCCGCCCCAGGACCCAAGGCCCC

  TGCCCCCTGGCAGCCCTGGACACGGTCCTCTGTGGGCGGTGGGGTCgGGG

  CTGTGGTGACGGTGGCATCGGGGAGCCTGTGCCCCCTCCCTGAAAGGGCG

  GAGAGGCTCAAGAGGGGACAGAAATGTCCTCCCCTAGGAAGACCTCGGAC

  GGGGGCGGGGGGGTGGTCTCCGACAGACAGATGCCCGGGACCGACAGACC

  TGCCGAGGGAAGAGGGCACCTCGGTCGGGTTAGGCTCCAGGCAGCACGAG

  GGAGCGAGGCTGGGAGGGTGAGGACATGGGAGCCTGAGGAGGAGCTGGAG

  ACTTCAGCAGGCCCCCAGCTCCGGGCTTCGGGCTCTGAGATGCTCGGACG

  CAAGGTGAGTGACCCCACCTGTGGCTGACCTGACCTCAgGGgGACAAGGC

  TCAGCCTGGGACTCTGTGTCCCCATCGCCTGcACAGGGGATTCCCCTGAT

  GGACACTGAGCCAACGACCTCCCGTCTCTCCCCGACCCCCAGGTCAGCCC

  AAgGCCaCTCCCACGGTCAACCTCTTCCCGCCCTCCTCTGAGGAGCTCGG

  CACCAACAAGGCCACCCTGGTGTGTCTAATAAGTGACTTCTACCCGGGCG

  CCGTGACGGTGACCTGGAAGGCAGGCGGCACCACCGTCACCCAGGGCGTG

  GAGACCACCAAGCCCTCGAAACAGAGCAACAACAAGTACGCGGCCAGCAG

  CTACCTGGCCCTGTCCGCCAGTGACTGGAAATCTTCCAGCGGCTTCACCT

  GCCAGGTCACCCACGAGGGGACCATTGTGGAGAAGACAGTGACGCCCTCC

  GAGTGCGCCTAGGTCCCTGGGCCCCCACCCTCAGGGGCCTGGAGCCACAG

  GACCCCCGCGAGGGTCTCCCCGCGACCCTGGTCCAGCCCAGCCCTTCCTC

  CTGCACCTGTCAACTCCCAATAAACCGTCCTCCTTGTCATTCAGAAATCA

  TGCTCTCCGCTCACTTGTGTCTACCCATTTTCGGGCTTGCATGGGGTCAT

  CCTCGAAGGTGGAGAGAGTCCCCCTTGGCCTTGGGgAAATCGAGGGGGGC

  GGGGGGAGGCCTGAGGCATGTGCCAGCGAGGGGGGTCACCTCCACGCCCC

  TGAGGACCTTCTAGAACCAGGGGCGTGGGGCCACCGCCAGAGTGGAAGGC

  TGTCCACTTTTCCCCCGGGCCCCCAGGCTCCCTCCTCCGTGTGGACCTTG

  TCCACCTCTGACTGGCCCAGCCACTCATGCATTGTTTCCCCGAAACCCCA

  GGACGATAGCTCAGCACGCGACAGTGTCCCCCTCTGAGGGCCTCTGTCCA

  TTTCAGGACGACCCGCATGTACAGCGTGACCACTCTGCTCACGCCCACTC

  ACCACGTCCTAGAGCCCCACCCCCAGCCCCATCCTTAGGGGCACAGCCAG

  CTCCGACCGCCCCGGGGACACCACCCTCTGCCCCTTCCCCAGGCCCTCCC

  TGTCACACGCACCACAGGGCCCTCCGTCCCGAGACCCTGCTCCCTCATCC

  CTCGGTCCCCTCAGGTAGCCTTCCACCCGCGTGTGTCCCGAGGTCCCAGA

  TGCAGCAAGGCCCCTGGGACAACGCCAGATCTCTGCTCTCCCCGACCCTC

  AGAAGCCAGCCCACGCCTGGCCCACCACCACTGCCTAACGTCCAAGTGTC

  CATAGGCTCGGGAcCTCcAaGTCCAGGTTCTGCCTCTGGGATTCCGCCAT

  GGGTCTGCCTGGAATGATGCACTTGGAGgAgCTCAGcATGGGATGcGGAA

  CTTGTCTAGcGCTCCTCAGATCCAcAGcTGCCTGtGAgAcacacacacac

  acacacacacaccAAAcaCGcATGCACGCACGCCGGCACACACGCTATTA

  CAGAGATGGCCACGGTAGCTGTGCCTCGAGGCCGAGTGGAGTGTCTAGAA

  CTCTCGGGGGTCCCCTCTGCAGACGACACTGCTCCATCCCCCCCGTGCCC

  TGAAGGGCTCCTCACTCTCCCATCAGGATCTCTCCAAGCTGCTGACCTGG

  AGAGGAAGGGGCCTGGGACAGGCGGGGACACTCAGACCTCCCTGCTGCCC

  CTCCTCTGCCTGGGCTTGGACGGCTCCCCCCTTCCCACGGGTGAAGGTGC

  AGGTGGGGAGAGGGCACCCCCCTCACCCTCCCAGACCCAGACCAGCCCCC

  GTGGCAGGGGCAGCCTGTGAGCCTCCAGCCAGATGCAGGTGGCCTGGGGT

  GGGGGGTGGAGGGGGCGGGAGGTTTATGTTTGAGGCTGTATTCATCTGTG

  TAATATttTCGGCGGTGGGACCCATCTGACCGTCCTCGGTGAGTCTCCCC

  TtttctttcctccttggggatccgagtgaaATcTGGGTCGATCTTCTCTC

  CGTTCTCCTCCGACTGGGGCTGAGGTCTGAACCTCGGTgGGGTCCGAAGA

  GGAGGCCCCTAGGCC*GGCTCcTCAGCCCCTCCAGCCCGACCCGCCCTCT

  TGACACAGGGTCCAGCTAAGGGCAGACAT***GGCTGCTAGTCCAGGGCC

  AGGCTcTGAGACCCAAGGGCGCTGCCCAAGGAACCCTTGCCCCAGGGACC

  CTGGGAGCAAAGCTCCTCACTCAGAGCCTGCAGCCCTGGgGTCTGAGGAC

  AAGGAGGGACTGAGGACTGGGCGTGGGGAGTTCAGGCgGGGACACCGGGT

  CCAGGGAGGTGACAAAGGCGCTGGGAGGGGGCGGACGGTGCCGGAGACTC

  CTCCTGGGCCCTGTGGGCTCGTGGTCCTTGTGAGGACCCTGAGGG*CTGA

  GGGGCCCCTGGGCCTAGGGACTTGCAGTGAGGGAGGCAGGGAGTGTCCCT

  TGAGAACGTGGCCTCCGCGGGCTGGGTCCCCCTCGTGCTCCCAGCAGGGA

  GGACACCCCAGAGCAAGCGCCCCAGGTGGGCGGGGAGGGTCTCCTCACAG

  GGGCAGCTGACAGATAGAC*GgccCCCGCCAGACAGATGCTTGATCCTGG

  TCag***TACTGGGTTCGCcACTTCCCTGAACAGGGGCCCTCCGAACAGA

  CACAGACGCAGACCaggCTCAGCACAgAAAATGCACTGACACCCAAAACC

  CTCATCTGggGGCCTGGCCGGCATCCCGCCCCAGGACCCAAGGCCCCTGC

  CCCCTGGCAGCCCTGGACACGGTCCTCTGTGGGCGGTGGGGTCgGGGCTG

  TGGTGACGGTGGCATCGGGGAGCCTGTGCCCCCTCCCTGAAAGGGCGGAG

  AGGCTCAAGAGGGGACAGAAATGTCCTCCCCTAGGAAGACCTCGGACGGG

  GGCGGGGGGGTGGTCTCCGACAGACAGATGCCCGGGACCGACAGACCTGC

  CGAGGGAAGAGGGCACCTCGGTCGGGTTAGGCTCCAGGCAGCACGAGGGA

  GCGAGGCTGGGAGGGTGAGGACATGGGAGCCTGAGGAGGAGCTGGAGACT

  TCAGCAGGCCCCCAGCTCCGGGCTTCGGGCTCTGAGATGCTCGGACGCAA

  GGTGAGTGACCCCACCTGTGGCTGACCTGACCTGACCtCAGGGGGACAAG

  GCTCAGCCTGGGACTCTgTGTCCCCATCGCCTGCACAGGGGATTCCCCTG

  ATGGACACTGAGCCAACGACCTCCCGTCTCTCCCCGACCCCCAGGTCAGC

  CCAAGGCCACTCCCACGGTCAACCTCTTCCCGCCCTCCTCTGAGGAGCTC

  GGCACCAACAAGGCCACCCTGGTGTGTCTA

  Seq ID No. 32

  GCCACGCCCACTCCATCATGCGGGGAGGGGATGGGCAGACCCTCCAGAAA

  GAAGCTCCCTGGGGTGCAGGTTAACAGCTTTCCCAGACACAGCCAGTACT

  AGAGTGAGGTGAATAAGACATCCTCCTTGCTTGTGAAATTTAGGAAGTGC

  CCCCAAACATCAGTCATTAAGATAAATAATATTGAATGCACTTTTTTTTT

  TTTATTTTTTTTTTTTGCTTTTTAGGGCCTAATCTGCAGCatatggaagt

  tcccaggctacaagtcgaaccagagctgcagctgccagcctacatcacag

  ccacagcaacaccagatccgagccacatctgtgactaacactgcagttca

  cagcaacgccagatccttaacccattgagtgaggccagggatcaaaccca

  catcctcatggatactagtctggttcgtaaaccactgagccaCAAGGGGA

  ACTCCTGAATGCAATATTTTTGAAAATTGAAATTAAATCTGTCACTCTTT

  CACTTAAGAGTCCCCTTAGATTGGGGAAAATTTAAATATCTGTCATCTTA

  GTGCATCTTTGCTCATATGATGTGAATAAAATCCCAAAATCCATATGAAT

  GAAGCATCAAAATGTACATGAAGTCAGCCTGACCCTGCACTGCCCTCACT

  TGCCTCATGTACCCCCCACCTCAAAGGAAGATGCAGAAAGGAGTCCAGCC

  CCTACACCGCCACCTGCCCCCACCACTGGAGCCCCTCAGGTCTCCCACCT

  CCTTTTCTGAGCTTCAGTCTTCCTGTGGCATTGCCTACCTCTACAGCTGC

  CCCCTACTAGGCCCTCCCCCTGGGGCTGAGCTCCAGGCACTGGACTGGGA

  AAGTTAGAGGTTAAAGCATGGAAAATTCCCAAAGCCACCAGTTCCAGGCT

  GCCCCCCACCCCACCGCCACGTCCAAAAAGGGGCATCTTCCCAGATCTCT

  GGCTGGTATTGGTAGGACCCAGGACATAGTCTTTATACCAATTCTGCTGT

  GTGTCTTAGGAAAGAaactctccctctctgtgcttcagtttcctcatcaa

  taaaAGGAGCAGGCCAGGTTGGAGGGTCTGTGACGTCTGCTGAAGCAGCA

  GGATTCTCTCTCCTTTTGCTGGAGGAGAACTGATCCTTCACCCCCAGGAT

  CAACAGAGAAGCCAAGGTCTTCAGCCTTCCTGGGGACCCCTCAGAGGGAA

  CTCAGGGCCACAGAGCCAGACCCTGATGCCAGAACCTTTGTCATATGCCC

  AGACGGAGACTTCATCCCCCTCCTCCTCAGACCCTCCAGGCCCCAACAGT

  GAGATGCTGAAGATATTAAGAGAAGGGCAAGTCAGcTTAAGTTTGGGGGT

  AGAGGGGAACAGGGAGTGAGGAGATCTGGCCTGAGAGATAGGAGCCCTGG

  TGGCCACAGGAGGACTCTTTGGGTCCTGTCGGATGGACACAGGGCGGCCC

  GGGGGCATGTTGGAGCCCGGCTGGTTCTTACCAGAGGCAGGGGGCACCCT

  CTGACACGGGAGCAGGGCATGTTCCATACATGACACACCCCTCTGCTCCA

  GGGCAGGTGGGTGGCGGCACAGAGGAGCCAGGGACTCTGAGCAAGGGGTC

  CACCAGTGGGGCAGTTGGATCCAGACTTCTCTGGGCCAGCGAGAGTCTAG

  CCCTCAGCCGTTCTCTGTCCAGGAGGGGGGTGGGGCAGGCCTGGGCGGCC

  AGAGCTCATCCCTCAAGGGTTCCCAGGGTCCTGCCAGACCCAGATTTCCG

  ACCGCAGCCACCACAAGAGGATGTGGTCTGCTGTGGCAGCTGCCAAGACC

  TTGCAGCAGGTGCAGGGTGGGGGGGTGGGGGCACCTGGGGGCAGCTGGGG

  TCACTGAGTTCAGGGAAAACCCCTTTTTTCCCCTAAACCTGGGGCCATCC

  CTAGGGGAAACCACAACTTCTGAGCCCTGGGCAGTGGCTGCTGGGAGGGA

  AGAGCTTCATCCTGGACCCTGGGGGGGAACCCAGCTCCAAAGGTGCAAGG

  GGCCCAGGTCCAAGGCTAGAGTGGGCCAAGCACCGCAATGGCCAGGGAGT

  GGGGGAGGTGGAGCTGGACTGGATCAGGGCCTCCTTGGGACTCCCTACAC

  CCTGTGTGACATGTTAGGGTACCCACACCCCATCACCAGTCAGGGCCTGG

  CCCATCTCCAGGGCCAGGGATGTGCATGTAAGTGTGTGTGAGTGTGTGTG

  TGTGGTGTAGTACACCCCTTGGCATCCGGTTCCGAGGCCTTGGGTTCCTC

  CAAAGTTGCTCTCTGAATTAGGTCAAACTGTGAGGTCCTGATCGCCATCA

  TCAACTTCGTTCTCCCCACCTCCCATCATTATCAAGAGCTGGGGAGGGTC

  TGGGATTTCTTCCCACCCACAAGCCAAAAGATAAGCCTGCTGGTGATGGC

  AGAAGACACAGGATCCTGGGTCAGAGACAAAGGCCAGTGTGTCACAGCGA

  GAGAGGCAGCCGGACTATCAGCTGTCACAGAGAGGCCTTAGTCCGCTGAA

  CTCAGGCCCCAGTGACTCCTGTTCCACTGGGCACTGGCCCCCCTCCACAG

  CGCCCCCAGGCCCCAGGGAGAGGCGTCACAGCTTAGAGATGGCCCTGCTG

  AACAGGGAACAAGAACAGGTGTGCCCCATCCAGCGCCCCAGGGGTGGGAC

  AGGTGGGCTGGATTTGGTGTGAAGCCCTTGAGCCCTGgAACCCAAcCACA

  GCAgGGCAGTTGGTAGATGCCATTTGGGGAGAGGCCCCAGGAGTAAGGGC

  CATGGGCCCTTGAGGGGGCCAGGAGCTGAGGACAGGGACAGAGACGGCCC

  AGGCAGAGGACAGGGCCATGAGGGGTGCACTGAGATGGCCACTGCCAGCA

  GGGGCAGCTGCCAACCCGTCCAGGGAACTTATTCAGCAGTCAGCTGGAGG

  TGCCATTGACCCTGAGGGCAGATGAAGCCCAGGCCAGGCTAGGTGGGCTG

  TGAAGACCCCAGGGGACAGAGCTCTGTCCCTGGGCAGCACTGGCCTCTCA

  TTCTGCAGGGCTTGACGGGATCCCAAGGCCTGCTGCCCCTGATGGTAGTG

  GCAGTACCGCCCAGAGCAGGACCCCAGCATGGAAACCCCAACGGGACGCA

  GCCTGCGGAGCCCACAAAACCAGTAAGGAGCCGAAGCAGTCATGGCACGG

  GGAGTGTGGACTTCCCTTTGATGGGGCCCAGGCATGAAGGACAGAATGGG

  ACAGCGGCCATGAGCAGAAAATCAGCCGGAGGGGATGGGCCTAGGCAGAC

  GCTGGCTTTATTTGAAGTGTTGGCATTTTGTCTGGTGTGTATTGTTGGTA

  TTGATTTTATTTTAGTATGTCAGTGACATACTGACATATTATGTAACGAC

  ATATTATTATGTGTTTTAAGAAGCACTCCAAGGGAACAGGCTGTCTGTAA

  TGTGTCCAGAGAAGAGAGCAAGAGCTTGGCTCAGTCTCCCCCAAGGAGGT

  CAGTTCCTCAACAGGGGTCCTAAATGTTTCCTGGAGCCAGGCCTGAATCA

  AGGGGgTCATATCTACACGTGGGGCAGACCCATGGACCATTTTCGGAGCA

  ATAAGATGGCAGGGAGGATACCAAGCTGGTCTTACAGATCCAGGGCTTTG

  ACCTGTGACGCGGGCGCTCCTCCAGGCAAAGGGAGAAGCCAGCAGGAAGC

  TTTCAGAACTGGGGAGAACAGGGTGCAGACCTCCAGGGTCTTGTACAACG

  CACCCTTTATCCTGGGGTCCAGGAGGGGTCACTGAGGGATTTAAGTGGGG

  GACCATCAGAACCAGGTTTGTGTTTTGGAAAAATGGCTCCAAAGCAGAGA

  CCAGTGTGAGGCCAGATTAGATGATGAAGAAGAGGCAGTGGAAAGTCGAT

  GGGTGGCCAGGTAGCAAGAGGGCCTATGGAGTTGGCAAGTGAATTTAAAG

  TGGTGGCACCAGAGGGCAGATGGGGAGGAGCAGGCACTGTCATGGACTGT

  CTATAGAAATCTAAAATGTATACCCTTTTTAGCAATATGCAGTGAGTCAT

  AAAAGAACACATATATATTTAAATTGTGTAATTCCACTTCTAAGGATTCA

  TCCCAAGGGGGGAAAATAATCAAAGATGTAACCAAAGGTTTACAAACAAG

  AACTCATCATTAATCTTCCTTGTTGTTATTTCAACGATATTATTATTATT

  ACTATTATTATTATTATTATTttgtctttttgcattttctagggccactc

  ccacggcatagagaggttcccaggctaggggtcaaatcggagctacagct

  gccggcctacgccagagccacagcaacgcaggatctgagccacagcaatg

  caggatctacaccacagctcatggtaacgctggatccttaacccaatgag

  tgaggccagggatcgaacctgtaacttcatggttcctagtcggattcatt

  aaccactgagccacgacaggaactccAACATTATTAATGATGGGAGAAAA

  CTGGAAGTAACCTAAATATCCAGCAGAAAGGGTGTGGCCAAATACAGCAT

  GGAGTAGCCATCATAAGGAATCTTACACAAGCCTCCAAAATTGTGTTTCT

  GAAATTGGGTTTAAAGTACGTTTGCATTTTAAAAAGCCTGCCAGAAAATA

  CAGAAAAATGTCTGTGATATGTCTCTGGCTGATAGGATTTTGCTTAGTTT

  TAATTTTGGCTTTATAATTTTCTATAGTTATGAAAATGTTCACAAGAAGA

  TATATTTCATTTTAGCTTCTAAAATAATTATAACACAGAAGTAATTTGTG

  CTTTAAAAAAATATTCAACACAGAAGTATATAAAGTAAAAATTGaggagt

  tcccatcgtggctcagtgattaacaaacccaactagtatccatgaggata

  tggatttgatccctggccttgctcagtgggttgaggatccagtgttgctg

  tgagctgtggtgtaggttgcagacacagcactctggcgttgctgtgactc

  tggcgtaggccggcagctacagctccatttggacccttagcctgggaacc

  tccatatgcctgagatacggcccTAAAAAGTCAAAAGCCAAAAAAATAGT

  AAAAATTGAGTGTTTCTACTTACCACCCCTGCCCACATCTTATGCTAAAA

  CCCGTTCTCCAGAGACAAACATCGTCAGGTGGGTCTATATATTTCCAGCC

  CTCCTCCTGTGTGTGTATGTCCGTAAAACACACACACACACACACACACG

  CACACACACACACACGTATCTAATTAGCATTGGTATTAGTTTTTCAAAAG

  GGAGGTCATGCTCTACCTTTTAGGCGGCAAATAGATTATTTAAACAAATC

  TGTTGACATTTTCTATATCAACCCATAAGATCTCCCATGTTCTTGGAAAG

  GCTTTGTAAGACATCAACATCTGGGTAAACCAGCATGGTTTTTAGGGGGT

  TGTGTGGATTTTTTTCATATTTTTTAGGGCACACCTGCAgcatatggagg

  ttcccaggctaggggttgaatcagagctgtagctgccggcctacaccaca

  gccacagcaacgccagatccttaacccactgagaaaggccagggattgaa

  cctgcatcctcatggATGCTGGTCAGATTTATTTCTGCTGAGCCACAACA

  GGAACTCCCTGAACCAGAATGCTTTTAACCATTCCACTTTGCATGGACAT

  TTAGATTGTTTCCATTTAAAAATACAAATTACAaggagttcccgtcgtgg

  ctcagtggtaacgaattggactaggaaccatgaggtttcgggttcgatcc

  ctggccttgctcggtgggttaaggatccagcattgatgtgagatatggtg

  taggtcgcagacgtggctcggatcccacgttgctgtggctctggcgtagg

  ccggcaacaacagctccgattcgacccctagccTGggaacctccatgtgc

  cacaggagcagccctaGAAAAGGCAAAAAGACAAAAAAATAAAAAATTAA

  AATGAAAAAATAAAATAAAAATACAAATTACAAGAGACGGCTACAAGGAA

  ATCCCCAAGTGTGTGCAAATGCCATATATGTATAAAATGTACTAGTGTCT

  CCTCGCGGGAAAGTTGCCTAAAAGTGGGTTGGCTGGACAGAGAGGACAGG

  CTTTGACATTCTCATAGGTAGTAGCAATGGGCTTCTCAAAATGCTGTTCC

  AGTTTACACTCACCATAGCAAATGACAGTGCCTCTTCCTCTCCACCCTTG

  CCAATAATGTGACAGGTGGATCTTTTTCTATTTTGTGTATCTGACAAGCA

  AAAAATGAGAACAggagttcctgtcgtggtgcagtggagacaaatctgac

  taggaaccatgaaatttcgggttcaatccctggcctcactcagtaggtaa

  aggatccagggttgcagtgagctgtggggtaggtcgcagacacagtgcaa

  atttggccctgttgtggctgtggtgtaggccggcagctatagctccaatt

  ggacccctagcctgggaacctccttatgccgtgggtgaggccctAAAAAA

  AAGAGTGCAAAAAAAAAAAATAAGAACAAAAATGATCATCGTTTAATTCT

  TTATTTGATCATTGGTGAAACTTATTTTCCTTTATATTTTTTATTGACTG

  ATTTTATTTCTCCTATGAATTTACCGGTCATAGTTTTGCCTGGGTGTTTT

  TACTCCGGTTTTAGTTTTGGTTGGTTGTATTTTCTTAGAGAGCTATAGAA

  ACTCTTCATCTATTTGGAATAGTAATTCCTCATTAAGTATTTGTGCTGCA

  AAAAATTTTCCCTGATCTGTTTTATGCTTTTGTTTGTGGGGTCTTTCACG

  AGAAAGCCTTTTTAGTTTTTACACCTCAGCTTGGTTGTTTTTCTTGATTG

  TGTCTGTAATCTGCGGCCAACATAGGAAACACATTTTTACTTTAGTGTTT

  TTTTCCTATTTTCTTCAAGTACGTCCATTGTTTTGGTGTCTGATTTTACT

  TTGCCTGGGGTTTGTTTTTGTGTGGCAGGAATATAAACTTATGTATTTTC

  CAAATGGAGAGCCAATGGTTGTATATTTGTTGAATTCAAATGCAACTTTA

  TCAAACACCAAATCATCGATTTATCACAACTCTTCTCTGGTTTATTGATC

  TAATGATCAATTCCTGTTCCACGCTGTTTTAATTATTTTAGCTTTGTGGA

  TTTTGGTGCCTGGTAGAGAACAAAGCCTCCATTATTTTCATTCAAAATAG

  TCCCGTCTATTATCTGCCATTGTTGTAGTATTAGACTTTAAAATCAATTT

  ACTGATTTTCAAAAGTTATTCCTTTGGTGATGTGGAATACTTTATACTTC

  ATAAGGTACATGGATTCATTTGTGGGGAATTGATGTCTTTGCTATTGTGG

  CCATTTGTCAAGTTGTGTAATATTTTACCCATGCCAACTTTGCATATTGT

  ATGTGAGTTTATTCCCAGGGTTTTTAATAGGATGTTTATTGAAGTTGTCA

  GTGTTTCCACAATTTCATCGCCTCAGTGCTTACTGTTTGCATAAAAGGAA

  ACCTACTCACTTTTGCCTATTGCTCTTGTATTCAATCATTTTAGTTAACT

  CTTGTGTTAATTTTGAGAGTTTTTCAGCTGACTGTCTGGGGTTTTCTTTA

  ATAGACTAGCCCTTTGTCTGTAAAGAATAATTTTATCGAATTTTTCTTAA

  CACTCACACTCTCCCCACCCCCACCCCCGCTCATCTCCTTTCATTGGGTC

  AAATCTGTAGAATACAATAAAAGTAAGAGTGGGAACCTTAGCCTTTAAGT

  CGATTTTGCCTTTAAATGTGAATGTTGCTATGTTTCGGGACATTCTCTTT

  ATCAAGTTGCGGATGTTTCCTTAGATAATTAACTTAATAAAAGACTGGAT

  GTTTGCTTTCTTCAAATCAGAATTGTGTTGAATTTATATTGCTATTCTGT

  TTAATTTTGTTTCAAAAAATTTACATGCACACCTTAAAGATAACCATGAC

  CAAATAGTCCTCCTGCTGAGAGAAAATGTTGGCCCCAATGCCACAGGTTA

  CCTCCCGACTCAGATAAACTACAATGGGAGATAAAATCAGATTTGGCAAA

  GCCTGTGGATTCTTGCCATAACTCTCAGAGCATGACTTGGGTGTTTTTTC

  CTTTTCTAAGTATTTTAATGGTATTTTTGTGTTACAATAGGAAATCTAGG

  ACACAGAGAGTGATTCAATGAGGGGAACGCATTCTGGGATGACTCTAGGC

  CTCTGGTTTGGGGAGAGCTCTATTGAAGTAAAGACAATGAGAGGAAGCAA

  GTTTGCAGGGAACTGTGAGGAATTTAGATGGGGAATGTTGGGTTTGAGGT

  TTCTATAGGGCACGCAAGCAGAGATGCACTCAGGAGGAAGAAGGAGCATA

  AATCTAGAGGCAAAAAGAGAGGTCAGGACTGGAAATAGAGATGCGAGACA

  CCAGGGTGGCAGTCAGAGAGCACAGTGTGGGTCAGAAGACAGTGGAAGAA

  CACAAGGGACAGAGAGGGATCTCCAACTTCACTGGGATGAGGGCCTTGTT

  GGCCTTGACCTGAGAGATTTCCAGGAGTTGAGGGTGGGAAGGAGAGGGCT

  CCTGCACATGTCCTGACATGAAACGGTGCCCAGCATATGGGTGCTTGGAA

  GACATTGTTGGACAGATGGATGGATGATGGATGATGGATGAATGGATGGA

  TGGAAGATGATGGATAAATGGATGATGGATGGATGGACAGAAGGACAAAG

  AGATGGACAGAAAGACAGTGATCTGAGAGAGCAGAGAAGGCTTCATGAAA

  GGACAGGAACTGAACTGTCTCAGTGGGTGGAGACAATGGTGTAGGGGGTT

  TCCACATGGAGGCACCAGGGGTCAGGAATAATCTAGTGTCCACAGGCCCA

  GGAAGGAAGCTGTCTGCAGGAAATTGTGGGGAAGAACCTCAGAGTCCTTA

  AATGAGGTCAGGAGTGGTCAGGAGGGTCTGATCAGGTAAGGACTCATGTC

  CATCATCACATGGTCACCTAAGGGCATGTAGCTCTCAGCATCTCCATCAG

  GACAGTCTCAGAATGGGGGCGGGGTCACACACTGGGTGACTCAAGGCGTG

  GGTCATGCCTGCCTCGGACGTGGGCCTGGGCATGGGGACACCTCCAGACC

  ATGGGCCCGCCCAGGGCTGCACTGGcctctggtgggctagctacccgtcc

  aagcaacacaggacacagccctacctgctgcaaccctgtgcccgaaacgc

  ccatctggttcctgctccagcccggccccagggaacaggactcaggtgct

  agcccaatggggttttgttcgagcctcagtcagcgtggTATTTCTCCGGC

  AGCGAGACTCAGTTCACCGCCTTAGGttaagtggttctcatgaatttcct

  agcagtcctgcactctgctatgccgggaaagtcacttttgtcgctggggg

  ctgtttccccgtgcccttggagaatcaaggattgcccaactttctctgtg

  ggggaggtggctggtcttggggtgaccagcaggaagggccccaaaagcag

  gagcagctgcctccagAATACAACTGTCGGCTACAGCTCAAACAGGAGGC

  CTGGACTGGGGTTTAACCACCAGGGCGGCACGAAGGAGCGAGGCTGGGAG

  GGTGAGGACATGGGAGCCTGAGGAGGAGCTGGAGACTTCAGCAGGCCCCC

  AGCTCCGGGCTTCGGGCTCTGAGATGCTCGGACGCAAGGTGAGTGACCCC

  ACCTGTGGCTGACCTGACCTCAGGGGGACAAGGCTCAGCCTGAGACTCTG

  TGTCCCCATCGCCTGCACAGgggattcccctgatggacactgagccaacg

  acctcccgtctctccccgacccccaggtcagcccaaggccgcccccacgg

  tcaacctcttcccgccctcctctgaggagctcggcaccaacaaggccacc

  ctggtgtgtctaataagtgacttctacccgAAGGGCGAATTCCAGCACAC

  TGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGATGCATAG

  CTTGAGTATCTA

  Seq ID No. 33

  agatctttaaaccaccgagcaaggccagggatcgaacccgcatcctcatg

  aatcctagttgggttcgttaaccgctgaaccacaatgggaactcctGTCT

  TTCACATTTAATTCACAACCTCTCCAGGATTCTGGGGGTGGGTGGGGAAT

  CCTAGGTACCCACTGGGAAAGTAATCCAAGGGGAGAGGCTCACGGACTcT

  AGGGATCGGCGGAGGAGGGAAGGTATCTCCCAGGAAACTGGCCAGGACAC

  ATTGGTCCTCCGCCCTCCCCTTCCTCCCACTCCTCCTCCAGACAGGACTG

  TGCCCACCCCCTGCCACCTTTCTGGCCAGAACTGTCCATGGCAGGTGACC

  TTCACATGAGCCCTTCCTCCCTGCCTGCCCTAGTGGGACCCTCCATACCT

  CCCCCTGGACCCCGTTGTCCTTTCTTTCCAGTGTGGCCCTGAGCATAACT

  GATGCCATCATGGGCTGCTGACCCACCCGGGACTGTGTTGTGCAGTGAGT

  CACTTCTCTGTCATCAGGGCTTTGTAATTGATAGATAGTGTTTCATCATC

  ATTAGGACCGGGTGGCCTCTATGCTCTGTTAGTCTCCAAACACTGATGAA

  AACCTTCGTTGGCATAGTCCCAGCTTCCTGTTGCCCATCCATAAATCTTG

  ACTTAGGGATGCACATCCTGTCTCCAAGCAACCACCCCTCCCCTAGGCTA

  ACTATAAAACTGTCCCAATGGCCCTTGTGTGGTGCAGAGTTCATGCTTCC

  AGATCATTTCTCTGCTAGATCCATATCTCACCTTGTAAGTCATCCTATAA

  TAAACTGATCCATTGATTATTTGCTTCTGTTTTTTCCATCTCAAAACAGC

  TTCTCAGTTCAGTTCGAATTTTTTATTCCCTCCATCCACCCATACTTTCC

  TCAGCCTGGGGAACCCTTGCCCCCAGTCCCATGCCCTTCCTCCCTCTCTG

  CCCAGCTCAGCACCTGCCCACCCTCACCCTTCCTGTCACTCCCTAGGACT

  GGACCATCCACTGGGGCCAGGACACTCCAGCAGCCTTGGCTTCATGGGCT

  CTGAAATCCATGGCCCATCTCTATTCCTCACTGGATGGCAGGTTCAGAGA

  TGTGAAAGGTCTAGGAGGAAGCCAGGAAGGAAACTGTTGCATGAAAGGCC

  GGCCTGATGGTTCAGTACTTAAATAATATGAGCTCTGAGCTCCCCAGGAA

  CCAAAGCATGGAGGGAGTATGTGCCTCAGAATCTCTCTGAGATTCAGCAA

  AGCCTTTGCTAGAGGGAAAATAGTGGCTCAACCTTGAGGGCCAGCATCTT

  GCACCACAGTTAAAAGTGGGTATTTGTTTTACCTGAGGCCTCAGCATTAT

  GGGAACCGGGCTCTGACACAAACACAGGTGCAGCCCGGCAGCCTCAGAAC

  ACAGCAACGACCACAAGCTGGGACAGCTGCCCCTGAACGGGGAGTCCACC

  ATGCTTCTGTCTCGGGTACCACCAGGTCACCATCCCTGGGGGAGGTAGTT

  CCATAGCAGTAGTCCCCTGATTTCGCCCCTCGGGCGTGTAGCCAGGCAAG

  CTCCTGCCTCTGGACCCAGGGTGGACCCTTGCTCCCCACTACCCTGCACA

  TGCCAGACAGTCAAGACCACTCCCACCTCTGTCTGAGGCCCCCTTGGGTG

  TCCCAGGGCCCCCGAGCTGTCCTCTACTCATGGTTCTTCCACCTGGGTAC

  AAAAGAGGCGAGGGACACTTTTCTCAGGTTTGCGGCTCAGAAAGGTACCT

  TCCTAGGGTTTGTCCACTGGGAGTCACCTCCCTTGCATCTCAATGTCAGT

  GGGGAAAACTGGGTCCCATGGGGGGATTAGTGCCACTGTGAGGCCCCTGA

  AGTCTGGGGCCTCTAGACACTATGATGATGAGGGATGTGGTGAAAAACCC

  CACCCCAGCCCTTCTTGCCGGGACCCTGGGCTGTGGCTCCCCCATTGCAC

  TTGGGGTCAGAGGGGTGGATGGTGGCTATGGTCAGGCATGTTTCCCATGA

  GCTGGGGGCACCCTGGGTGACTTTCTCCTGTGAATCCTGAATTAGCAGCT

  ATAACAAATTGCCCAAACTCTTAGGCTTAAAACAACACACATTTATTCCT

  CTGGGTCCCAGGGTCAGAAGTCCAAAATGAGTCCTATAGGCTAAATTTGA

  GGTGTCTCTGGGTTGAGCTCCTCCTGGAAGCCTTTTCCAGCCTCTAGAGT

  CCCAAGTCCTTGGCTCTGGGCCCCTCCCTCAAGCTTCAAAGCCACAGAAG

  CTTCTAATCTCTCTCCCTTCCCCTCTGACCTCTGCTCCCATCCTCATACC

  CTGTCCCCTCACTCTGACCCTCCTGCCTCCCTCTTTCCCTTATAAAGACC

  CTGCATGGGGCCACGGAGATAATCCAGGGTAATCGCCCCTCTTCCAGCCC

  TTAACTCCATCCCATCTGCAAAATCCCTGTCACCCCATAATGGACCTACT

  GATGGTCTGGGGGTTAGGACGTGGACAACTTGGGGCCTTATTCATCTGAT

  CACAACTCCAGTTCCCAGACCCCCAGACCCCCGGGCATTAGGGAAACTTC

  TCCCAGTTCCTCTCCCTCTGTGTCCTGCCCAGTCTCCAGGATGGGCCACT

  CCCGAGGGCCCTTCAGCTCAGGCTCCCCCTCCTTTCTCCCTGGCCTCTTG

  TGGCCCCATCTCCTCCTCCGCTCACAGGGAGAGAACTTTGATTTCAGCTT

  TGGCTCTGGGGCTTTGCTTCCTTCTGGCCATTGGCTGAAGGGCGGGTTTC

  TCCAGGTCTTACCTGTCAGTCATCAAACCGCCCTTGGAGGAAGACCCTAA

  TATGATCCTTACCCTACAGATGGAGACTCGAGGCCCAGAGATCCTGAGTG

  ACCTGCTCACATTCACAGCAGGGACTGAACCCCAGTCACCTACCCAACTC

  CAGGGCTCAGCGCTTTTTTTTTTTTTTTCTTTTTgccttttcgagggccg

  ctcccgcaacatatggagatttccaggctaggggtctaattggagcagtc

  gacactggcctaagccaaagccacagcaacaagggcaagccgcttctgca

  gcctataccacagctcacggcaatgccggatccttaacccactgagcaaa

  gccagggattgaacctgcaacctcatgtttcctagtcaaatttgttaacc

  actgacccatgacgggaactcccAGGGCTCAGCTCTTGACTCCAGGTTCG

  CAGCTGCCCTCAAAGCAATGCAACCCTGGCTGGCCCCGCCTCATGCATCC

  GGCCTCCTCCCCAAAGAGCTCTGAGCCCACCTGGGCCTAGGTCCTCCTCC

  CTGGGACTCATGGCCTAAGGGTACAGAGTTACTGGGGCTGATGAAGGGAC

  CAATGGGGACAGGGGCCTCAAATCAAAGTGGCTGTCTCTCTCATGTCCCT

  TCCTCTCCTCAGGGTCCAAAATCAGGGTCAGGGCCCCAGGGCAGGGGCTG

  AGAGGGCCTCTTTCTGAAGGCCCTGTCTCAGTGCAGGTTATGGGGGTCTG

  GGGGAGGGTCAATGCAGGGCTCACCCTTCAGTGCCCCAAAGCCTAGAGAG

  TGAGTGCCTGCCAGTGGCTTCCCAGGCCCAATCCCTTGACTGCCTGGGAA

  TGCTCAAATGCAGGAACTGTCACAACACCTTCAGTCAGGGGCTGCTCTGG

  GAGGAAAAACACTCAGAATTGGGGGTTCAGGGAAGGCCCAGTGCCAAGCA

  TAGCAGGAGCTCAGGTGGCTGCAGATGGTGTGAACCCCAGGAGCAGGATG

  GCCGGCACTCCCCCCAGACCCTCCAGAGCCCCAGGTTGGCTGCCCTCTTC

  ACTGCCGACACCCCTGGGTCCACTTCTGCCCTTTCCCACCTAAAACCTTT

  AGGGCTCCCACTTTCTCCCAAATGTGAGACATCACCACGGCTCCCAGGGA

  GTGTCCAGAAGGGCATCTGGCTGAGAGGTCCTGACATCTGGGAGCCTCAG

  GCCCCACAATGGACAGACGCCCTGCCAGGATGCTGCTGCAGGGCTGTTAG

  CTAGGCGGGGTGGAGATGGGGTACTTTGCCTCTCAGAGGCCCCGGCCCCA

  CCATGAAACCTCAGTGACACCCCATTTCCCTGAGTTCACATACCTGTATC

  CTACTCCAGTCACCTTCCCCACGAACCCCTGGGAGCCCAGGATGATGCTG

  GGGCTGGAGCCACGACCAGCCCACGAGTGATCCAGCTCTGCCAATCAGCA

  GTCATTTCCCAAGTGTTCCAGCCCTGCCAGGTCCCACTACAGCAGTAATG

  GAGGCCCCAGACACCAGTCCAGCAGTTAGAGGGCTGGACTAGCACCAGCT

  TTCAAGCCTCAGCATCTCAAGGTGAATGGCCAGTGCCCCTCCCCGTGGCC

  ATCACAGGATCGCAGATATGACCCTAGGGGAAGAAATATCCTGGGAGTAA

  GGAAGTGCCCATACTCAAGGATGGCCCCTCTGTGACCTAACCTGTCCCTG

  AGGATTGTACTTCCAGGCGTTAAAACAGTAGAACGCCTGCCTGTGAACCC

  CCGCCAAGGGACTGCTTGGGGAGGCCCCCTAAACCAGAACACAGGCACTC

  CAGCAGGACCTCTGAACTCTGACCACCCTCAGCAAGTGGCACCCCCCGCA

  GCTTCCAAGGCAC

  Seq ID No. 34

  AACAAGATGCTACCCCACCAACAAAATTCACCGGAGAAGACAAGGACAGG

  GGGTTCCTGGGGTCCTGACAGGGTCACCAAAGAGGGTTCTGGGGCAGCAG

  CAACTCCAGCCGCCTCAGAACAGAGCCTGGAAGCTGTACCCTCAGAGCAG

  AGGCGGAGAGAGAAAGGGCCTCTTGGTGGGTCAGCAGGAGCAGAGGCTCA

  GAGGTGGGGGTTGCAGCCCCCCCTTCAACAGGCCAACACAGTGAAGCAGC

  TGACCCCTCCACCTTGGAGACCCCAGACTCCTGTCTCCCACGCCACCTTG

  GTTTTTAAGGTAATTTTTATTTTATATCAGAGTATGGTTGACTTACAATG

  TTGTGTTGGTTTCAGGTGTACAGCAGAGTGATTCACTTCTACATAGACTC

  ATATCTATTCTTTCTCAGATTCTTTTCCCATATAGGTTATTACAGAATAT

  TGAGTAGATCCCTGCTGATTACCCATTTTTATAATTGTATATGTTAATCC

  CAAACTCCTAATTTATCCCTCCCCAGACTATGATTCTTTATATCTCTATC

  TGTTTCCTAATCTGTCTCCTCTAAGTCACCCTAGGAGAGCAGAGGGGTCA

  CGTCTGTCCTGTCCTGGCCCAGCCACCTCTCTCCACCCAGGAATCCCTTG

  CATTTGGTGCCAAGGGCCCGGCCCCGCCCTAAAGAGAAAGGAGAACGGGA

  TGTGGACAGGACACCGGGCAGAGAGGGACAAGCAGAGGATGCCAGGGTAG

  GGAGGTCTCCAGGGTGGATGGTGGTCTGTCCGCAGGCAGGATGAGGCAGG

  AAGGGTGTGGATGTACTCGGTGAGGCTGGCGCATGGCCTGGAGTGTCCTG

  AGCCCTGGGAGGCCTCAGCCCTGGATCAGATCTGTGATTCCAAAGGGCCA

  CTGCATCCAGAGACCGTTGAGTGGCCCATTGTCCTGAACCATTTATAGAA

  CACAGGACAAGCGGTACCTGACTAAGCTGCTCACAGATTCCATGAGGCTG

  ATGCCAGGGTTGTCACCCCATCTCACAGGCAGGGAAACTGATGCATATAC

  TGCAGAGCCAGGCAGAGGCCCTCCCAGTGCCCCCTCCCAGCCTGTGGCCC

  CCCTCCAGTGGCTGGACACTGAGGCCACACTGGGGCACCCTGTGGAGATC

  t

  Seq ID No. 35

  AGATCTGGCCAGGCCAGAGAAGCCCATGTGGTGACCTCCCTCCATCACTC

  CACGCCCTGACCTGCCAGGGAGCAGAAAGTAGGCCCAGGGTGGACCCGGT

  GGCCACCTGCCACCCCATGGCTGGGAGAAGGGAGGGCCTGGGCAAAGGGC

  CTGGGAAGCCTGTGGTGGGACCCCAGACCCCAGGGTGGACAGGGAGGGTC

  CCACACCCACAGCCATTTGCTTCCCTCTGTGGGTTCAGTGTCCTCATCTC

  ATCTGTGGGGAGGGGGCTGATAATGAATCTCCCCCATTGGGGTGGGCTTG

  GGGATTAAAGGGCCAGTGTCTGTGATATGCCTGGACCATAGTGACCCTCA

  CCCTCCCCAGCCATTGCTGTCACCTTCCGGGCTCTTGCCCAGGCCTGCCT

  GACATGCTGTGTGACCCTGGGCAAGATGATCCCCCTTTCTGGGCCCCAGC

  CTTCCTCTCTGCTCCGGAAGTGCTTCCTGGGGAAACCTGTGGGCTGGATC

  CTATAGGAAACCTGTCCAATTCCTGGATGCACAGAGGGGCAGGGAGGCCC

  TGGGCCTGGAGGGGCAGGGAGGCTCGAGGTGGGAGCAGGGTAGGGGCCAG

  TCCAGGGCAAGGAGGTGGGTGGGTAGGGTG

  Seq ID No. 36

  GATCTGTGTTCCATCTCAGAGCTATCTTAGCAGAGAGGTGCAGGGGCCTC

  CAGGGCCACCAAAGTCCAGGCTCAGCCAGAGGCAATGGGGTATCGATGAG

  CTACAGGACACAGGCGTCAGCCCAGTGTCAGGGAGAATCACCTTGTTTGT

  TTTCTGAGTTCCTCTTAAAATAGAGTTAATTGGTCTTGGCCTTACGGTTT

  ACAATAACAACTGCACCCTGTAAACAACGTGAAGAGTACAGAACAACAAA

  TGGGGGAAAACATATTTCACCTGAAAGAGCCACCGCTCATATTTTGATGG

  ATTTCCTTCTAGTTTAATCCTGTTTTAATTGTAAACTGTTAAAACAAACA

  TAAATAAAGAAAATGCATCTGTAAAGTTTAAAAGTCATATCTATGGTGAT

  GGTTGCAAAACACTGTGAATGTTCACTTTGAAATCGTGAACTCTACGTGA

  TATGCATGTCCCGTTAATTAACCTCACAGGCTCAGAATGTGGTTCATTAT

  TTCTTTAATTTTCCTTTAATTTTATGTCCTCTGTGTGTGCCCTTAAACCA

  ACTACTTTTCAGCTCTGCCTGTTTTTGACCTTCACATAGATGACATTTGT

  GAGTGTTTTCTTTCTCAACACTGGGTCTGATACCCACCCACGCTGTCTGC

  TGTCACTGCGGACGTGGAGGGCCACCACCCAGCTATGGCCCCAGCCAGGC

  CAACACTGGATGAATCTGCCCCCAGAGCAGGGCCACCAACACTGGAGGTG

  CAGAGAGGGTTTCTTCAGGGCCATCATTATCCAAGGCATTGTTTCTACTG

  TAAGCTTTCAAAATGCTTCCCCTGATTATTAAAAGAAATAATAAGATGGG

  GGGAAAGTACAAGAAGGGAAGTTTCCAGCCCAGCCTGAAGATCGTGCTGG

  TTGTATCTGGAGCCTGTCTTCCTGACAGGCCTCTATTCCCAGAGTTA

  Seq ID No. 37

  GGATCCTAGGGAAGGGAGGGCGGGGGCCTGGACAAAGGGGGCCTAAAGGA

  CATTCTCACCTATCCCACTGGACCcctgctgtgctctgagggagggagca

  gagagggggtctgaggccttttcccagCTCCTCTGAGTCCCTCCTCCGAG

  CACCTGGACGGAAGCCCCTCCTCAGGGAGTCCTCAGACCCCTCCCCTCCA

  GCCAGGTTGGCCTGTGTGGAGTCCCCAGTAAGAATAGAATGCTCAGGGCT

  TCGAGCTGAGCCCTGGCTACTTGGGGGGGTGCTGGGGATTGGGGGTGCTG

  GGCGGGGAGCTGGGGTGTCACTAGATGCCAGTAGGCTGTGGGCTCGGGTC

  TGGGGGGTCTGCACATGTGCAGCTGTGGGAAGGCCCTATTGGTGGTACCC

  TCAGACACATATGGCCCCTCAATTTCTGAGACCAGAGACCCCAGTCTGGC

  CTTCCCAGAACAGCTGCCCCTGGTGGGGGAGATGTAGGGGGGCCTTCAGC

  CCAGGACCCCCAACGGCAGGGCCTGAGGCCCCCATCCCCTTGTCCTGGGC

  CCAGAGCCTCAGCTATCAGGCCTATCAGAGATCCTGGCTGCCCAGCTCAG

  GTTCCCCAGGAGCCAGAGGGAGGCCAGGGGTTACTAGGAAATCCGGAAAG

  GGTCTTTGAGGCTGGGCCCCACCCTCTCAGCTTTCACAGGAGAAACAGAG

  GCCCACAGGGGGCAAAGGACTTGCCAGACTCACAATGAGCCCAGCAGCTG

  GACTCAAGGCCCAGTGTTCGGCCCCACAACAGCACTCACGTGCCCTTGAT

  CGTGAGGGGCCCCCTCTCAGCCAGGCATTCAGACCTGTGACCTGCATCTA

  AGATTCAGCATCAGCCATTCTGAGCTGAAGAGCCCTCAGGGTCTGCAGTC

  AAGGCCACAGGGCCAGACCTCCAACGGCCAGACATCCCAGCCAGATTCCT

  TTCTGGTCAATGGGCCCCAGTCTGGCTTGGCTCCTGCAGGCCCAGTGCCG

  CCTTCTTCCCCTGGGCCTGTGGAGTCCAGCCTTTCAGTTTCCCACCCACA

  TCCTCAGCCACAATCCAGGCTCAGAGGCAATGTCCGTGGGCAGCCCCTGT

  GTGACCCCTCTGTGGGTGATCCTCAGTCCTACCCTTAGCAGACAGCGCAT

  GAGGGGCCCTCTTGAACCTGAGGGATACTCCATGTCGGAGGGGAGAAGCT

  GGCCTTCCCCACCCCCACTTCCAGGCCTTGGGGAGCAGAGAAAGACCCCA

  GACCTGGGTCCCTTCTAACAGGCCAGGCCCCAGCCCAGCTCTCCACCAGC

  CCCAGGGGCCTCGGGTCCACGCCTGGGGACTGGAGGGTGGGCCTGTCAGG

  CGCTGACCCAGAGGCAGGACAGCCAAGTTCAGGATCCCAGCCAGGTGGTC

  CCCGTGCACCATGCAGGGGTGTCACCCACACAGGGGTGTTGCCACCCTCA

  CCTGACTGTCCTCATGGGCCACATGGAGGTATCCTGGGTTCATTACTGGT

  CAACATACCCGTGTCCCTGCAGTGCCCCCTCTGGcgcacgcgtgcacgcg

  cacacgcacacactcatacaGAGGCTCCAGCCAACAGTGCCCTCTAGTAG

  GCACTGCTGTCACTTCTCTAAAAGGTCGCAATCATACTTGTAAAGACCCA

  AGATTGTTCAGAAATCCCAGATGGAGAAGTCTGGAAAGATCtTTTTCTCC

  TTTCACGGGCTGGGGAAATGTGACCTGGCCAAGGTCACACAGCAAGTGGT

  GGAACCCTGGCCCCTGATTCCAGCTCATTCCAGTTCCCAAGGCCCTGCCA

  GAGCCCAGAGGCTGGGCCCTCTGGGGCAGAGGAGCTGGGGTCCTCCCCCC

  TACACAGAGCACACAGCCCCGCAAGAGAGAAGAGACACCTTGGGGAGAGG

  AATCTCCAGACCAGAGATCCCAGTATGGGTCTCCTCTATGCTGACGGGAT

  GGGATGTCAAGAGGGGAGGGGGCTGGGCTTTAGGGAAACACACAAAAATC

  GCTGAGAACACTGACAGGTGCGACACACCCACCCCTAATGCTAACCTGTG

  GCCCATTACTCAgatct

  Seq ID No. 38

  GATCTTCTCCTAAGACCAAGGAAAACTGGTCATACCAGGTCCACTTGTCC

  CCTGTGGCCATTGTCCCTCCTTCCCCAGAAGAAACAAGCACTTTCCACTC

  CACAAGTAGCTCCTGATCAGCTTGGAAGCCCGGTGCTGCTCTGGGCCCTG

  GGGACACGGCAGGGGCATCAGAGACCAAATCCTGGAACAAAGTTCCAGTG

  GGTGAGGCAGGCCGGACAAGCAACACGTTATACCATAATATGAGGCAAAA

  TATAATGTGAGTTCTTTATGAAAGGAAGGGGTTGCAGGTGCAACTGTTGG

  CTTAGGTGGATGGTCACCCCTGAATGGAGGAGGGGGTTCCCAGGGCATGT

  GCCTGGGGAGAAGGGCTCCTGGCAGGAGGGACAGCAAGTGCAAGGGCCCT

  GTGATCAAATGTGCCTGGCAAGTTGCAGGAACAGCTAGAAGGCCAGCAAG

  GTTGGAACCAAGGAAGGGGTCAGGGGAGGGGCAGGGCCCTCAGGGCCTTG

  CCCAGCAGCCTGAGCATCTGGAGATTTGTCCAAAGTTTCAAATGTACCTG

  GGCAACCTCATGCCCATATACCATTCCTAACTTCTGCACTTAACATCTCT

  AGGACTGGGACCCAGCCAGTCAAGCGGGGGGACCCAGAGAGCTCCGGTGT

  GAACACCGAGGTGCTGGTGGGTCTGCGTGTGTGGACATAGGGCAGTCCCG

  GTCCTTCCTTCACTAACACGGCCCGGGAAGCCCTGTGCCTCCCTGGTGCG

  CGGGTCGGCGCTTCCGGAGGGTACAGGCCCACCTGGAGCCCGGGCACAGT

  GCATGCAAGTCGGGTTCACGGCAACCTGAGCTGGCTCTGCAGGGCAGTGG

  GACTCACAGCCAGGGGTACAGGGCAGACCGGTCCTGCCTCTGCGCCCCTC

  CCTGGCCTGTGGCCCCTGGACGTGATCCCCAACAGTTAGCATGCCCCGCC

  GGTGCTGAGAACCTGGACGAGGTCCGCAGGCGTCACTGGGCGGTCACTGA

  GCCCGCCCCAGGCCCCCTCTGCCCCTTCCTGGGGTGACCGTGGACTCCTG

  GATGACCCTGGACCCTAGACTTCCCAGGGTGTCTCGCGGAGGTTCCTCAG

  CCAGGATCTCTGCGTCTCCTCCTTCCATAGAGGGGACGGCGCCCCCTTGT

  GGCCAAGGAGGGGACGGTGGGTCCCGGAGCTGGGGCGGAGAACACAGGGA

  GCCCCTCCCAGACCCCGCTCTGGGCAGAACCTGGGAAGGGATGTGGCCAT

  CGGGGGATCCCTCCAGGCCATCTCCTCAGATGGGGGCTGGTCGACTAGCT

  TCTGAGTCCTCCAAGGAACCGGGTCCTTCTAGTCATGACTCTGCCCAGAT

  GAAGAAGGAGAGCACTTCTCTCCATCAGGAGGATCTGAGCTTCTCTTAAT

  TAGAATCAGCTCCTTGGCTTCTACCCCTTAAAAAAAGGTACAGAAACTTT

  GCACCTTGATCCAGTATCAGGGGAATTTATCAATCAATGTGGGAGAAATT

  GGCATCTTTACCACACTGAATCTTTCAATCCATGAATATCCTCTCTCTCT

  TCCATGCATAGGTTTTAATAATTCTCAATGGAGTTTAATGTAAGTTTTCC

  TCATAGACAATTGCCTTTGGACATCTCTTTAGACTCATCTCTAGTAAACT

  GATATTCTTAATGCAATTATAAAATGTATCCTGCTTAATGTTATTTTCTA

  TTCATTTGCTGTTATATAGAGATACAATGAGTTTCCACATTTGAAACTGG

  ATCTGGTAAATTGGCTACCCTTTTTTTATAGATTCTATTAATTTTTATAC

  ATTCTGTGGGACTTGCTACATACTTAATCATGTCACCTGTGAAGAATGAC

  AATTTGGTTGCTACCCTCCCAATTCTTATATGTCTCATTTCTTTCCCTCT

  GCTGGTACTCTGGCAGCAGCAGGGAAGATAATGGGCCTCCTTATCTTGTC

  ACAAAAGGATGTTTTTAAAGATTTCGTTATAAAACATAACGCTTTCTGGT

  TTTCTTTAAAGATTCTCTCACCAGCTTAAGAAAATTTTCTTATACTCTGT

  ATGATAAATGGGTTTTTGACAATCATTTGTTGCATTTTACCTAGTGTTTT

  CTCTGCATCTTTATATGCTTTTTCTCCTTTAATCCTGAAAATTGTTTCGA

  TTTTTCTAACATTGAACCAATCTTACATTCCTGGAATGGATGGACCAGAC

  TAGTCCACATGTTTATTCTGCCCAATGGCTAGATTTTGTGTTCaatattt

  tgttcagaatgtttgcatctatattcttGAGTGAGACAGAGCTGCCCTTG

  TTAGGTTTCACAACCGAGGTTGTGTTAGCTTCATAAAATGAGACGTTTAT

  TCTCTAAAAGAATTGTTTCGCTTCTCTGGATGAATTTGTGTAAGGTTAGA

  ATTGCTTACCAGTGAagatctCGGGgCCAGTTCTTCTTTAGGGGAAGATT

  TTCAACAATTAAGCTCAATGCCTTTAGAAGAACTGAGAGTTTCTATTATT

  TCTTGAGTTAAATATATGTATTTAATTAGACTTTCTAGGAATAGTCTCAT

  TTCATCTCAAATAATTGACATATGCTATTAAAGCAGATTCTCATGAACCA

  TTGTAGGTATTCCAGGTCTAGAAAAATGTTCCCCTTTGCATCCCTAATGT

  GTTTAATTTTCACCTTCTTTCTTTTGTTCTTGAGAAATTCACCAAATCAT

  TTTCAATTTCAGTCATATCCCAAAGCAACCAACTCTCTACCTTCTTGTTT

  TATCATCCCTGCTGGATTTTTGTTATCTACTTCTTCAGTATTTGTTCTTC

  CCTTTCTTCTATTCCTCATTCCATTTTTCCCTTGTTTTCTAACTTTCTGA

  GATATATGCTTAGTTCCTTCATTTGAAGCCTTTTTATTTTCTTTTTTTTT

  TTTTGGTCTTTTTGTCTTTtGTTGTTGTTGTTGTGCTATTtCTTGGGCCG

  CTCCCGCGGCATATGGAGGTTCCCAGGCTAGGAGTCGAATCGGAGCTGTA

  GCCACCGGCCTACGCCAGAGCCACAGCAATGCGGGATCCGAGCCGCGTCT

  GCAACCTACACCACAGCTCATGGCAACGCCGGATCGTTAACCCACTGAGC

  AAGGGCAGGAACCGAACCCGCAACCTCATGGTTCCTAGTCGGATTCGTAA

  CCACTGTGCCACAACAGGAACTCCGCCTTTTATTTTTCTATAAAAATTTC

  TATGTACATTTTAAGGTTATAGGTTTCCTTCTATGTACCCCATTGGCTGT

  ATCCTCAGGGTTCTGTGGAGTGATTTCATTATTGTTCAAGTTCAATATGT

  CTTCTGATTTTCCAATTTGAATACCTCTCTAAATCAGTAGGTGAATATTT

  CTTTTTCTTTTTCTTTTCTTTTCTTCTTTTTTTTTTTCTTTCAGCCAGGT

  CCATGGCATGCAGAAATTCCCAGGCCAGGAATCAAACTCTCACCATGGCA

  GTGACAATGTCGGATCCTTTACCCACTAGGCCACCAGGGAACTCTGGGAG

  CATATGTTTTTATTTCCCGACATCTGAGGATGCCTAGTATGTCTTCATTA

  TTGATTTCTAGTTTGCCACTGATTTCTAGTATTTTGCTCATAGAGTGTAT

  GCTCAATGGTTTTGGTCATTTGAAATGTATTTAGTCCTGCTTTATGACCC

  AGTATGTGGTCAGTTTTGTCAATGTTCCTTTTCTGCTTGAAGAGAACCTA

  CATGCTGTAACTCTGGGTGCATGTTCTGTATATAAGTCTATAGGCTGAGC

  CGGGGGAGCCTTCTAATCTGCCGTTATCTTCTTCGAGTTATTCTAGGTAC

  TATTTCTTAGCCATAAACCTTTAAATTCTGATATCAATATAATGACCCCA

  GCCCGCTTAGGGTCGGCACTTCATGTTATCTTTTTCCATCCATTTAATCC

  CTCCCCACTGTTTTGGCCACACCCGTGGGATATGGGAGTTCCTGGGCCAA

  GGATCaGATCTGAGCCGCAGCTGCCACCTATGCCACAGCAgcagcaatga

  tggatctttaacccactgcaccacactggggattgaacccaagcctcagc

  agcaacccaagctactgcagagacaacaccagatccttaacctgctgtgc

  catagcgggaaTTTCCATCCATTTACTTTCAAGCCAGCTGAATAACCTAG

  CCCACCATGCCTGGACATGGGTGCTCTGCTTCAAATGATTTTGTTCAGTC

  AGCATCCATCTCTGAAATGTGTGCCAAGCATTTATATGCATGCAAGAGTC

  ATGTTGGCACTTCTATCATTTCCAACAGTTCAGTAGCCTTTGTATCATGA

  CATTTCTTGGCCTTTTCTCTACAATATTTGAGGCTGAGCAGACTGGCCGT

  GCCCCTGTCCATGCTTCCAGAGCCTGTGTGCAGACTTCTGCTCTAGACAG

  AGACAGCTAACCATCCTGCAGTGCCCAGAAAACCCAACTCAAAGACCCTC

  AAGTAAGGAAGGATTTATTGGCTCACGTAATCTGGAATCCAGGCATGGGG

  TATTCAGGGCCACCTGAACCAGAGGCCCTGGCCCTGTTCTCTAAGCTTCT

  TCCTGCCCTGCCCTCGTTCTGGAAGTGACCCTGAAGGACAGCAATGAAGG

  GCAGCTCCCCCAGGGACAGATGACTGAGAGGTCCATTTCAAGTCCAACTT

  GGCCTAGATTGAGAGGCAGCAAGAAATATGGACCTACAGTGAGTCACAGG

  ATTTACCAGTGGTTTGGCTGGGTTGTCAGTGTTACAGGCTAAACATTTGG

  GTCCCTCCAAAATTAACATGTTGCCACTCTAACCACCAAAATCatggtat

  ttgggggtggggcccttggaggtaattaggtttagaaAGAATGAAGAGGG

  GGCCCTTGTGATGGGACTAGTGCCTTTATAGAGAGAGAAGAGAGAGGG

  Seq ID No. 39

  CACCTCATCCCCAACCACCTGGATGGTGGCAAGTGGCAGGCTGAGAGGCT

  GCATATGAGCTCATCAAGAGGGTCCCCACCCCACAGAGGCTGACCCAGCT

  GCCACTGCCACCTAGTGGCTGATCGGCCAAGAGCAGGAGCCCCAGGGGCA

  GCTCCATTCCCTGGGGCGGCCAGGGAACCACCTGGTGGTAGGACAATTCC

  ATTGCACCTCATCCATCAGGAAAAGGTTTGCCTTCCCTGGCAGTAATGCA

  TCTTCCCATAACATGGTCCCTGGCCTCTTGGAATGGCTTGGCCACCGTCA

  TGGCCTCACCCACAAAGCCTTGTGTCTCAGCAAGGAACTTATTCCACAGC

  AAAGGACTTGCAGCCTGGAATGAACTGGTCTGACTACATACCCCATTGCC

  CAGAAGTAGGTGGTCTATTGCAAAGTGGAGTGGCTTACCCAAGACTCAGT

  TGTGCCCAAGTTGAGAGATAGCATCCTAAAATATGGGCTTATGTCTCACT

  GGCTGAGGTTTATTCTTTGAATCAAAGACAATTATATGGTGTGGTCCCCC

  CAGAGATAGAATACATGAGTCTGGGAATCAAGGGATAGAAGTAAGAAGAG

  ATTTTGTCACCATTAATCCCAATAACTCGCCCAAAGAATATTTGCTTTCT

  GTCCTGGCAGCTCTGCTGCTTTGGCAATAACTTCCTAGAATATAATGTCT

  CCACCAGGGGACTCCACAACGGTTCCATTGATTTGAAGCCAATGGGCAGA

  GGAGGGGCTGCCTTACTGGTCGGACTGGTCAGCCCTGATTACTAAGGAGA

  AATCAGGCAACTTCAACAAAACTAAGGCAGGGGGGACTTTGTCTAGAACC

  CAAAGCACTAAGCATCTTAGTACTTTTTAGTTCTCAGAGCCTCCAAGAAC

  AAAGATTTAGCCCCTCAGCACCACCAGGTAAAGAACAGGTAAATCCAGCT

  GAGGACAAGAGAAATATTGAATGGATAGAGGAAGAAAGAAATTATAGATA

  TCAACTATGGCCTCATGACTAGAGTCTCCAGATTAAGCGGAATAAAAATA

  CAGATGATTaGATCTGAACATCAGGCCAAACAACGAACAACAGTTTAAGT

  GCGACCTAGGCAATATTTGGGACATACTTATACTAAAATTTTTTCGCTAT

  TTGAGCATCCTGTATTTTATCTGGCAACTTTATTCATCCCTAGCGAAAAA

  GGAACTGTGGTAACTTAGTGTATTTTTACTTTGCTCATTATTGTGTATAT

  ACCTACTTGTATTTATCAATCATATTTACTCTGTTCTCAGTATTACTTTA

  TATAGCAGTTGGTGGTGATGGTTAGCAACATATTCAGTGGAACTGTGACT

  GAATTTGAGGAGAAATTAACAGAGTTGGCTGTGGCTACAATAACCCTTCG

  GGACATGTGTCCCCTCATTTTGGGGAGATGGTTagatctCTGGGTAAATG

  TTAGGGCATCTGAGCCAGAAACCAAGATTTTGCCAGCTGGTGCAATGTCA

  GATTTTACCAGCAGAGGGTGCCAGAGGAATGCGGCAAAACCCGAGTGCCA

  GAAAGCACCTCCCTGTTTTCCAGCTTTTCTTCCTTTTTATTTATTTTATT

  TACGGCCCAGGAGTCCGTAATAGCGCTGAGGATGGCCCAGGCTCTTCTCA

  GCAGCCCTGACTGACTAGTTCAGCAATGCGCTCAGGCCCCATCTGGCCAC

  CGGGCAGCCTCTTCTGTGGTAGCTCCAGCCTCAGCCAGTGCAAAAGGCTA

  CCCTACACTGGCGCCACTTCTACAATCAGCACTGGCCACACCCTCCACGC

  CATCCGGCACGGAGCCAGGTGATCTGCCGGCCAGATTGCAGTTCGTGCTG

  CCTGAGTCCAGGTGATTACACTGGCTGCATCTTTTCTTTCTGGACCAtTC

  attccattttttt

Bovine Lambda Light Chain
• In a further embodiment, nucleic acid sequences are provided that encode bovine lambda light chain locus, which can include at least one joining region-constant region pair and/or at least one variable region, for example, as represented by Seq ID No. 31. In Seq ID No 31, bovine lambda C can be found at residues 993-1333, a J to C pair can be found at the complement of residues 33848-35628 where C is the complement of 33848-34328 and J is the complement of 35599-35628, V regions can be found at (or in the complement of) residues 10676-10728, 11092-11446, 15088-15381. 25239-25528, 29784-30228, and 51718-52357. Seq ID No. 31 can be found in Genbank ACCESSION No. AC117274. Further provided are vectors and/or targeting constructs that contain all or part of Seq ID No. 31, for example at least 100, 250, 500, 1000, 2000, 5000, 10000, 20000, 500000, 75000 or 100000 contiguous nucleotides of Seq ID No. 31, as well as cells and animals that contain a disrupted bovine lambda gene.

• Seq ID No 31

  1	tgggttctat gccacccagc ttggtctctg atggtcactt gaggccccca tctcatggca
  61	aagagggaac tggattgcag atgagggacc gtgggcagac atcagaggga cacagaaccc
  121	tcaaggctgg ggaccagagt cagagggcca ggaagggctg gggaccttgg gtctagggat
  181	ccgggtcagg gactcggcaa aggtggaggg ctccccaagg cctccatggg gcggacctgc
  241	agatcctggg ccggccaggg acccagggaa agtgcaaggg gaagacgggg gaggagaagg
  301	tgctgaactc agaactgggg aaagagatag gaggtcagga tgcaggggac acggactcct
  361	gagtctgcag gacacactcc tcagaagcag gagtccctga agaagcagag agacaggtac
  421	cagggcagga aacctccaga cccaagaaga ctcagagagg aacctgagct cagatctgcg
  481	gatgggggga ccgaggacag gcagacaggc tccccctcga ccagcacaga ggctccaagg
  541	gacacagact tggagaccaa cggacgcctt cgggcaaagg ctcgaacaca catgtcagct
  601	caaaatatac ctggactgac tcacaggagg ccagggaggc cacatcatcc actcagggga
  661	cagactgcca gccccaggca gaccccatca accgtcagac gggcaggcaa ggagagtgag
  721	ggtcagatgt ctgtgtggga aaccaagaac cagggagtct caggacagcg ctggcagggg
  781	tccaggctca ggctttccca ggaagatggg gaggtgcctg agaaaacccc acccaccttc
  841	cctggcacag gccctctggc tcacagtggt gcctggactc ggggtcctgc tgggctctca
  901	aaggatcctg tgtccccctg tgacacagac tcaggggctc ccatgacggg caccagacct
  961	ctgattgtgg tcttcttccc ctcgcccact ttgcaggtca gcccaagtcc acaccctcgg
  1021	tcaccctgtt cccgccctcc aaggaggagc tcagcaccaa caaggccacc ctggtgtgtc
  1081	tcatcagcga cttctacccg ggtagcgtga ccgtggtcta gaaggcagac ggcagcacca
  1141	tcacccgcaa cgtggagacc acccgggcct ccaaacagag caacagcaag tacgcggcca
  1201	gcagctacct gagcctgatg ggcagcgact ggaaatcgaa aggcagttac agctgcgagg
  1261	tcacgcacga ggggagcacc gtgacgaaga cagtgaagcc tcagagtgtt cttagggccc
  1321	tgggccccca ccccggaaag ttctaccctc ccaccctggt tccccctagc ccttcctcct
  1381	gcacacaatc agctcttaat aaaatgtcct cattgtcatt cagaaatgaa tgctctctgc
  1441	tcatttttgt tgatacattt ggtgccctga gctcagttat cttcaaagga aacaaatcct
  1501	cttagccttt gggaatcagg agagagggtg gaagcttggg ggtttgggga gggatgattt
  1561	cactgtcatc cagaatcccc cagagaacat tctggaacag gggatggggc cactgcagga
  1621	gtggaagtct gtccaccctc cccatcagcc gccatgcttc ctcctctgtg tggaccgtgt
  1681	ccagctctga tggtcacggc aacacactct ggttgccacg ggcccagggc agtatctcgg
  1741	ctccctccac tgggtgctca gcaatcacat ctggaagctg ctcctgctca agcggccctc
  1801	tgtccactta gatgatgacc cccctgaagt catgcgtgtt ttggctgaaa ccccaccctg
  1861	gtgattccca gtcgtcacag ccaagactcc ccccgactcg acctttccaa gggcactacc
  1921	ctctgcccct cccccagggc tccccctcac agtcttcagg ggaccggcaa gcccccaacc
  1981	ctggtcactc atctcacagt tcccccaggt cgccctcctc ccacttgcat ggcaggaggg
  2041	tcccagctga cttcgaggtc tctgaccagc ccagctctgc tctgcgaccc cttaaaactc
  2101	agcccaccac ggagcccagc accatctcag gtccaagtgg ccgttttggt tgatgggttc
  2161	cgtgagctca agcccagaat caggttaggg aggtcgtggc gtggtcatct ctgaccttgg
  2221	gtggtttctt aggagctcag aatgggagct gatacacgga taggctgtgc taggcactcc
  2281	cacgggacca cacgtgagca ccgttagaca cacacacaca cacacacaca cacacacaca
  2341	cacacacgag tcactacaaa cacggccatg ttggttggac gcatctctag gaccagaggc
  2401	gcttccagaa tccgccatgg cctcactctg cggagaccac agctccatcc cctccgggct
  2461	gaaaaccgtc tcctcaccct cccaccgggg tgacccccaa agctgctcac gaggagcccc
  2521	cacctcctcc aggagaagtt ccctgggacc cggtgtgaca cccagccgtc cctcctgccc
  2581	ctcccccgcc tggagatggc cggcgcccca tttcccaggg gtgaactcac aggacgggag
  2641	gggtcgctcc cctcacccgc ccggagggtc aaccagcccc tttgaccagg aggggggcgg
  2701	acctggggct ccgagtgcag ctgcaggcgg gcccccgggg gtggcggggc tggcggcagg
  2761	gtttatgctg gaggctgtgt cactgtgcgt gtttgctcgg tggagggacc cagctggcca
  2821	tccggggtga gtctcccctt tccagctttc cggagtcagg agtgacaaat gggtagattc
  2881	ttgtgttttt cttacccatc tggggctgag gtctccgtca ccctaggcct gtaaccctcc
  2941	cccttttagc ctgttccctc tgggcttctt cacgtttcct tgagggacag tttcactgtc
  3001	acccagcaaa gcccagagaa tatccagatg gggcaggcaa tatgggacgg caagctagtc
  3061	caccctctta ccttgggctc cccgcggcct ccggataatg tctgagctgc ctccctggat
  3121	gcttcacctt ctgagactgt gaggcaagaa accccctccc caaaagggag gagacccgac
  3181	cccagtgcag atgaacgtgc tgtgagggga ccctgggagt aagtggggtc tggcggggac
  3241	cgtgatcatt gcagactgat gccccaggca gggtgagagg tcatggccgc cgacaccagc
  3301	agctgcaggg agcacaggcc gggggcaagt catgcagaca ggacaggacg tgtgaccctg
  3361	aagagtcaga gtgacacgcg gggggggggc ccggagctcc cgagattagg gcttgggtcc
  3421	taacgggatc caggagggtc cacgggccca ccccagccct ctccctgcac ccaatcaact
  3481	tgcaataaaa cgtcctctat tgtcttacaa aaaccctgct ctctgctcat gtttttcctt
  3541	gccccgcatt taatcgtcaa cctctccagg attctggaac tggggtgggg nnnnnnnnnn
  3601	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  3661	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn agcttatgtg gtgggcaggg gggtagtaag
  3721	atcaaaagtg cttaaattaa taaagccggc atgatatacg agtttggata aaaaatagat
  3781	ggaaaagtaa gaaaggacag gaggggggtg aggcggaaga aagggggaag aaggaaaaaa
  3841	aaataagaga gaggaacaaa gaaagggagg ggggccggtg atgggggtgg gatagaatat
  3901	aataattgga gtaaagagta gcgggtggct gttaattccg ggggggaata gagaaaaaaa
  3961	aaaaaaaatg tgcgggtggg cggtaagtat ggagatttta taaatattat gtgtggaata
  4021	atgagcgggg gtggacgggc aaggcgagag taaaaagggg cgagagaaaa aaattaggat
  4081	ggaatatatg gggtaaattt taaatagagg gtgatatatg ttagattgag caagatataa
  4141	atatagatgg tgggggaaaa gagacaaggg tgagcgccaa aacgccctcc cgtatcattt
  4201	gccttccttc ctttaccacc tcgttcaaac tctttttcga gaaccctgaa gcggtcaggc
  4261	ccggggctgg gggtgggata cccggggagg ggctgcgcct cctcctttgc agagggggtc
  4321	gaggagtggg agctgaggca ggagactggc aggctggaga gatggctgtt gacttcctgc
  4381	ctgtttgaac tcacagtcac agtgccagac ccactgaatt gggctaaata ccatattttt
  4441	ctggggagag agtgtagagc gagcgactga ggcgagctca tgtcatctac agggccgcca
  4501	gctgcaggga ctttgtgtgt gtcgtgctcg ttgctcagtt gtgtccgact ctttatgact
  4561	tcatggactg taacctgcca ggctcctctg tccgtggaat tctccaggca agaatactgg
  4621	agtgggtagc cattctcatc tccgggggat cttcctgacc caagaatcaa acctgagtct
  4681	cccgcattgc aggcagcttc tttcttgtct gagccaccag ggaagcccct taagtggagg
  4741	atctaaatag agtgtttagg agtataagag aaaggaagga cgtctataca agatccttcg
  4801	gttcctgtaa ctacgactcg agttaacaag ccctgtgtga gtgagttgcc agtaattatt
  4861	gctaacctgt ttctttcact cactgagcca ggtatcctgt gagacggcat acttacctcc
  4921	tcttctgcat tcctcgggat ggagctgtgc ggtggcctct aggactacca catcgaccag
  4981	gtcagaccca gggacagagg attgctgaga tgcactgaga agtttgtcag cctaggtctt
  5041	cacccacaca gactgtgctg tcgtctacca cgtaattctt cctgtccaaa gaactggtta
  5101	aacgctcctg aagcgtattc tggtctgctt caaaaagtgc ctctttcctt tataagttcc
  5161	gccaatcctg gactttgtcc caggccagtc tactttattt gtgggaaagg tttttttggt
  5221	cttttttgtt ttaaactctg cagaaattgc ttacactttt ggtgtgcaat ggctcactct
  5281	tacggttcta gctgtattca aaggggttgc ttttctttgt ttttaaagct ttttgaacgt
  5341	ggaccatttt taaagtcttt attaaacgtc taacatcgtt tctggtttat tttctggtgg
  5401	tctggccatg aggcctacgg gtcttagctc ccctaccagg gtccaaccca catcccttgc
  5461	actggacggc aaggtcttaa cctttgaacc accagagagc ttctgaaagg ggctgctttt
  5521	ctccaatcct ctttgctccc tgcctgctgg tagggattca gcacccctgc aatagccctg
  5581	tctgttctta ggggctcagt agcctttctg cctgggtgtg gagctggggt tgtaagagag
  5641	cttcatggat ttggacacga cctacgactc agaggtaaga ctccatctta gcgctgtaat
  5701	gacctctttc caacaaccac ccccaccacc ctggaccact gatcaggaga gatgattctc
  5761	tctcttatca tcaacgtggt cagtcccaaa cttgcacccg gcctgtcata gatgtagcag
  5821	gtaagcaata aatatttgtt gaatgttaag tgaattgaaa taacataagt gaaaaagaaa
  5881	acacttaaaa acatgtgttt ttataattac acagtaaaca tataatcatt gtagaaaaaa
  5941	atcgaaagag tggcgggggc caagtgaaaa ccaccatccc tggtatgtcc acccgcccgg
  6001	gtagccccag gtaagaggtg cggacacgga tggccctgta gacacagaga cacacgctca
  6061	tatgctgggt cttgtcttgt gacctcttgg ggatgatgtt attttcacga tgccattcaa
  6121	accttctacc acaccatttt tagagggtcg ttcatcgtaa atcagttcac tgctttgttt
  6181	tctgattttg aaagtgtcac attcttcgag aaatgagaag gaacaggcgc gcataaggaa
  6241	gaaagtaaac acgtggcctt gcttccaggg ggcactcagc gtgttggtgt gcacgctggc
  6301	agtcttttct ctgtgacagt catggccttt tcccaaaggt gggctcagat aagaccgcct
  6361	cccatcccct gtccctgtcc ccgtccccta cggtggaacc cacccacggc acgtctccga
  6421	ggccctttgg ggctgtggac gttaggctgt gtggacatgc tgctggtggg gacccagggc
  6481	tgggcagcac gttgtccctg ggtcccgggc cagtgaggag ctcccaagga gcagggctgc
  6541	tgggccaaag ggcagtgcgt cccgaggcca tggacaaggg gatacatttc ctgctgaagg
  6601	gctggactgc gtctccctgg ggccccttgg agtcatgggc agtggggagg cctctgctca
  6661	ccccgttgcc cacccatggc tcagtctgca gccaggagcg cctggggctg ggacgccgag
  6721	gccggagccc ctccctgctg tgctgacggg ctcggtgacc ctgccgcccc ctccctgggg
  6781	ccctgctgac cgcgggggcc accccggcca gttctgagat tcccctgggg tccagccctc
  6841	caggatccca ggacccagga tggcaaggat gttgaggagg cagctagggg gcagcatcag
  6901	gcccagaccg gggctgggca ggggctgggc gcaggcgggt gggggggtct gcacnccccc
  6961	acctgcnagc tgcncnnncn tttgntnncg tcctccctgn tcctggtctg tcccgcccgg
  7021	ggggcccccc ctggtcttgt ttgttccccc tccccgtccc ttcccccctt tttccgtcct
  7081	cctcccttct tttattcgcc ccttgtggtc gttttttttc cgtccctctt ttgttttttt
  7141	gtctttttct ttttccccct cttctccctt gctctctttt tcattcgtcg gtttttctgc
  7201	tcccttccct ctcccccccg ctttttttcc ctgtctgctt tttgtgttct ccctctctac
  7261	cccccctgca gcctattttt tttatatatc catttccccc tagtatttgg cccccgctta
  7321	cttctcccta atttttattt tcctttcttt aactaaaatc accgtgtggt tataagtttt
  7381	aacctttttt gcaccgccca caatgcaatc ttcacgcacg ccccccccgt cagcctcctt
  7441	aaataccttt gcctactgcc cccctccttg tataataacg cgtcacgtgg tcaaccatta
  7501	tcacctctcc accaccttac cacattttcc ttcnnnnnnn nnnnnnnnnn nnnnnnnnnn
  7561	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  7621	nnnnnnnnnn nnntgaaaaa agaaaaggct gggcaggttt taatatgggg gggttggagt
  7681	ggaatgaaaa tgcattggag tggttgcaac aaatggaaag gtctcaggag cgctcctccc
  7741	ccatcaggag ctggaaagaa gtggaagcaa agcaaggaat tcgtgtgatg gccagaggtc
  7801	aggggcaggg agctgcaaag actgccggct gtttgtgact gnccgtctcc gggtgcattt
  7861	gttagcaggg aggcattaca ctcatgtctt ggtttgctaa ctaattctta ctattgttta
  7921	gttgcaaggt catgtctgac tctttgcaac ccagggactg cagcccgcca ggctcctctg
  7981	tccatgggat ttcgcaggca agaatactgg aggtggtagc cattttcttc accatgggat
  8041	cttcccgagc cagaaatgga acccgagtcg cctcctgtgc atggggtctg ctgcctaaca
  8101	ggcagatatt tgacgtctga gccaacaggg aggacagacg gtaattatac caaccattga
  8161	aagaggaatt acacactaat ctttatcaaa atctttcaaa cagtagagga gaaaggatac
  8221	tctctagttt attccataaa gttggaatta cgcttatcaa taaagacatt acaagaaaag
  8281	aaagtgaagc cccaaatgcc ttataaatat acaagaaaaa atcttttaag atattagcca
  8341	acttaatcaa caaaaaatgt atcaaaagtc caagtaacat tcaccccagg aatgcaagtg
  8401	tggttcagcc taagacaatc agtcatgagt ataccacgga aacaaattaa agagaaaaga
  8461	cattaaatct cacaaatggt gcagaaaaag atttggcaat atcgaacatc ttttcatgac
  8521	caaaggaaaa aaaagaaaca aaacaccaga aaattctgtg tagaaagaat atatctcaac
  8581	ccaatgaagg gcatttatga aaaacccaca gcatacatca cactccatga gaaagactga
  8641	aagctttccc cactgccatt gaactctgtc ctggaaattc tagtcacagc gacagaacaa
  8701	gagaaagaaa taacggccgt ctaaactggt aggaagaaat caaagcgtct ctattctctg
  8761	ggcgcataat acaatataga caaatttcta aagtccacaa aaattcctag agctcataat
  8821	gaatccagaa atgcgtcagg gctcaagatt cagatgcaaa aatcgtctgg gttttgatgc
  8881	accaacaaac aattccatta acaataatac caaggaatta atttaactta gaagagaaaa
  8941	gacctgttta cagagagtta taaaacattt ggtgatgaaa ttaaataaga gtaaatcata
  9001	tagaaacacc gttcgtgttt tggagaccta atgtcataaa cgtggcaaca cagagacgcc
  9061	tcacggggaa ccctgagcct ccttctccaa acaggcctgc tcatcatttc acaggtaacc
  9121	tgagacccta aagcttgact ctgaggcact ttgagggcat gaagagagca gtagctcctc
  9181	ccatgggacc gacagtcaag gcccagggaa tgaccacctg gacagatgac ttcccggcct
  9241	catcagcagt cggtgcagag tggccaccag ggggcagcag agagtcgctc aacactgcac
  9301	ctggagatga ggcaacctgg gcatcaggtg cccatgcagg ggctggatac ccacacctca
  9361	cacctgagga caggggccgg ctttctgtgg tgtcgccctc tcaggatgca cagactccac
  9421	cctcttcgct tgcattgaca gcctctgtcc ttcctggagg acaagctcca ccttccccat
  9481	ctctccccag ggggctgggg ccaacagtgt tctctcttgt ccactccagg aacacagagc
  9541	caagagattt atttgtctta attagaaaaa ctatttgtat tcctgcattt ccccagtaac
  9601	tgaaggcaac tttaaaaaat gtatttcctg gacttccctg gtgggccagt ggctagactc
  9661	tgagctccca gtgcatgggg cctgggttca atccctgctc aggaaactac atcccacagg
  9721	ctgcaaataa gatcctgcat gccacccgat gcaggcaaag aaacaagtgt tcggtatgca
  9781	tgtatttcac gtgaggtgtt tctataattt acagccagta ttctgtctta cacttagtca
  9841	ttcctttgag cacatgatcg gtcgatggcc cagaccacac acaggaatac tgaggcccag
  9901	cacccaccgg ctgcccagaa cctcatggcc aagggtggac acttacagga cctcagggga
  9961	cctttaagaa cgccccgtgc tcttggcagc ggagcagtgt taagcatggc tctgtccctc
  10021	gggagctgtg tctgggctgc gtgcatcacc tgtggtgtgg gcctggtgag ggtcaccgtc
  10081	caggggccct cgagggtcag aagaaccttc ccttaaaagt tctagaggtg gagctagaac
  10141	cagacccaca tgtgaactgc acccaaaaac agtgaaggat gagacacttc aaagtcctgg
  10201	gtgaaattaa gggccttccc ctgaaccagg atggagcaga ggaaggactt ggcttccagg
  10261	aaaccctgac gtctccaccg tgactctggc cggggtcatg gcagggccca ggatcctttg
  10321	gtgcaaagga ctcagggttc ctggaaaata cagtctccac ctctgagccc tcagtgagaa
  10381	gggcttctct cccaggagtg gggcaaggac ccagattggg gtggagctgt ccccccagac
  10441	cctgagacca gcaggtgcag gagcagcccc gggctgaggg gagtgtgagg gacgttcccc
  10501	ccgctctcaa ccgctgtagc cctgggctga gcctctccga ccacggctgc aggcagcccc
  10561	caccccaccc cccgaccctg gctcggactg atttgtatcc ccagcagcaa ggggataaga
  10621	caggcctggg aggagccctg cccagcctgg gtttggcgag cagactcagg gcgcctccac
  10681	catggcctgg accccctcct cctcggcctc ctggctcact gcacaggtga gccccagggt
  10741	ccacccaccc cagcccagaa ctcggggaca ggcctggccc tgactctgag ctcagtggga
  10801	tctgcccgtg agggcaggag gctcctgggg ctgctgcagg gtgggcagct ggaggggctg
  10861	aaatccccct ctgtgctcac tgctaggtca gccctgaggg ctgtgcctgc cagggaaagg
  10921	ggggtctcct ttactcagag actccatcca ccaggcacat gagccggggg tgctgagact
  10981	gacggggagg gtgtccctgg gggccagaga atctttggca cttaatctgc atcaggcagg
  11041	gggcttctgt tcctaggttc ttcacgtcca gctacctctc ctttcctctc ctgcaggcgc
  11101	tgtgtcctcc tacgagctga ctcagtcacc cccggcatcg atgtccccag gacagacggc
  11161	caggatcacg tgttgggggc ccagcgttgg aggtganaat gttgagtggc accagcagaa
  11221	gccaggccag gcctgtgcgc tggtctccta tggtgacgat aaccgaccca cgggggtccc
  11281	tgaccagttc tctggcgcca actcagggaa catggccacc ctgcccatca gcggggcccg
  11341	ggccaaggat gaggccgact attactgtca gctgtgggac agcagcagta acaatcctca
  11401	cagtgacaca ggcagacggg aagggagatg caaaccccct gcctggcccg cgcggcccag
  11461	cctcctcgga gcagctgcag gtcccgctga ggcccggtgc cctctgtgct cagggcctct
  11521	gttcatcttg ctgagcagcg gcaagtgggc attggttcca agtcctgggg gcatatcagc
  11581	acccttgagc cagagggtta ggggttaggg ttagggttag gctgtcctga gtcctaggac
  11641	agccgtgtcc cctgtccatg ctcagcttct ctcaggactg gtgggaagat tccagaacca
  11701	ggcaggaaac cgtcagtcgc ttgtggccgc tgagtcaggc agccattctg gtcagcctac
  11761	cggatcgtcc agcactgaga cccggggcct ccctggaggg caggaggtgg gactgcagcc
  11821	cggcccccac accgtcaccc caaaccctcg gagaaccgcg ctccccagga cgcctgcccc
  11881	tttgcaacct gacatccgaa cattttcatc agaacttctg caaaatattc acaccgctcc
  11941	tttatgcaca ttcctcagaa gctaaaagtt atcatggctt gctaaccact ctccttaaat
  12001	attcttctct aacgtccatc ttccctgctc cttagacgcg ttttcattcc acatgtctta
  12061	ctgcctttgg tctgctcgtg tattttcttt tttttttttt ttttattgga atatatttgc
  12121	gttacaatgt tgaatttgaa ttggtttctg ttgtacaaca atgtgaatta gttatacatg
  12181	tcctgaggag gggcggctgc gtgggtgcag gagggccgag aggagctact ccacgttcaa
  12241	ggtcaggagg ggcggccgtg aggagatacc cctcgtccaa ggtaagagaa acccaagtaa
  12301	gacggtaggt gttgcgagag ggcatcagag ggcagacaca ctgaaaccat aatcacagaa
  12361	actagccaat gtgatcacac ggaccacagc ctggtctaac tcagtgaaac taagccatgc
  12421	ccatggggcc aaccaagatg ggcgggtcat gtgcccatgg ggccaaccaa gatgggcggg
  12481	tcatggtgaa gaggtctgat ggaatgtggt ccactggaga agggaaaggc aaaccacttc
  12541	agtattcttg ccttgagagc cccatgaaca gtatgaaaag gcaaaatgat aggatactga
  12601	aagaggaact ccccaggtca gtaggtgccc aatatgctac tggagatcag tggagaaata
  12661	actccagaaa gaatgaaggg atggagccaa agcaaaaaca atacccagtt gtggatgtga
  12721	ctggtgatag aagcaagggc caatgatgta aagagcaata ttgcatagga acctggaatg
  12781	ttaagtccaa gannnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  12841	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnagaatttt
  12901	gagcattact ttactagcgt gtgagacgag tgcaattgtg cggtagtttg agcattcttt
  12961	ggcattgcct ttctttggga ttggaatgaa aactgacctg ttccaggcct gtggccactg
  13021	ctgagttttc caaatttgct ggcgtattga gtgcatcact ttaacagcat catcttttag
  13081	gatttgaaat agctcaactg gaattctatc actttagcta attccattca ttagctttgt
  13141	ttgtagtgat gcttcctaag gcccccctgg ctttatcttc ctggatgtct ggctctggtg
  13201	agtgatcaca ccgctgtgat tatctgggtc atgaaggtct ttttgtatag ttcttcttag
  13261	gaacagatat tatgatctcc atccttgcat ctcgttatat ctagagaagc actgactccc
  13321	ttcatggtga cgtcagatcc tcatgactaa caaatggcct tttgtaagat gagtgcctca
  13381	tggtattgag ctcccccgtc accaagacct tatgactgac ctcccccact gccccaggtg
  13441	cctctcgaag cgtctgagat gccgcctccc aggctgcact cctcattttg cccccaataa
  13501	aacttaactt gcagctctcc agctgtgcat ctgtgtttag ttgacagtac aaatataatg
  13561	gaaaatttaa attaaatata atctatgggg agaaatccaa acatcttatg agggagagag
  13621	agggagagaa aggaaagaag aagaagcagg aggaggagga gagtagagaa acagggggag
  13681	ggcggcaggg agacagaggg gaggacaccg aggggaaagg gaggaaggcg agtgcagtga
  13741	gagagaggcc agagttcatc agagtctgga ctcgcagccc aatcccacgg gtgtgtcccg
  13801	aagcagggga gagcctgagc caggcggaga cagagctgtg tctccagtcc tcgtggccgt
  13861	gacctggagc tgtgtggtca gcccccctga ccccagcctg gccctgctgg tggtcggagg
  13921	cagtgatcct ggacacagtg tctgagcgtc tgtctgaaat ccctgtggag gcgccactca
  13981	ggacggacct cgcctggccc cacctggatc tgcaggtcca ggcccgagtg gggcttcctg
  14041	cctggaactg agcagctgga ggggcgtctg caccccagca gtggagcggc cccaggggcg
  14101	ctcagagctg ccggggggac acagagcttg tctgagaccc agggctcgtc tccgaggggt
  14161	cccctaaggt gtcttctggc cagggtcaga gccgggatga gcacaggtct gagtcagact
  14221	ttcagagctg gtggctgcat ccctggggac agagggctgg gtcctaacct gggggtcaga
  14281	gggcaggacg ggagcccagc tgacccctgg ggactggcct cctctgtggt ctcccctggg
  14341	cagtcacagc ttccccggac gtggactctg aggaggacag ctggggcctg gctgtcagga
  14401	gggggttcga gaggccacac tcagaggagg agaccctggc ctgcttgggt tgtgactgag
  14461	tttttggggt cctctaggag actctggccc tgcaggccct gcaaggtcat ctctagtgga
  14521	gcaggactcc acaagattga tgaactgaat cctctaggag aggtgtggtt gtgagggggc
  14581	agcattctag aaccaacagc gtgtgcaggt agctggcacc gggtctagtg gcggcgggca
  14641	gggcactcag ggccgactag gggtctgggg gattcaatgg tgcccacagc actgggtctt
  14701	ccatcagaat cccagacttc acaaggcagt ttcggggatt aggtcaggac gtgagggcca
  14761	cagagaggtg gtgatggcct agacaagtcc ttcacagaga gagctccagg ggccatgata
  14821	agatggatgg gtctgtattg tcagtttccc cacatcaaca ccgtggtccc gccagcccat
  14881	aatgctctgt ggatgcccct gtgcagagcc tacctggagg cccgggaggc ggggccgcct
  14941	gggggctcag ctccggggta accgggccag gcctgtccct gctgtgtcca cagtcctccc
  15001	ggggttggag gagagtgtga gcaggacagg agggtttgtg tctcacttcc ctggctgtct
  15061	gtgtcactgg gaacattgta actgccactg gcccacgaca gacagtaata gtcggcttca
  15121	tcctcggcac ggaccccact gatggtcaag atggctgttt tgccggagct ggagccagag
  15181	aactggtcag ggatccctga gcgccgctta ctgtctttat aaatgaccag cttaggggcc
  15241	tggcccggct tctgctggta ccactgagta tattgttcat ccagcagctc ccccgagcag
  15301	gtgatcttgg ccgtctgtcc caaggccact gacactgaag tcaactgtgt cagttcatag
  15361	gagaccacgg agcctggaag agaggaggga gaggggatga gaaggaagga ctccttcccc
  15421	aagtgagaag ggcgcctccc ctgaggttgt gtctgggctg agctctgggt ttgaggcagg
  15481	ctcagtcctg agtgctgggg gaccagggcc ggggtgcagt gctggggggc cgcacctgtg
  15541	cagagagtga ggaggggcag caggagaggg gtccaggcca tggtggacgt gccccgagct
  15601	ctgcctctga gcccccagca gtgctgggct ctctgagacc ctttattccc tctcagagct
  15661	ttgcaggggc cagtgagggt ttgggtttat gcaaattcac cccccggggg cccctcactc
  15721	agaggcgggg tcaccacacc atcagccctg tctgtcccca gcttcctcct cggcttctca
  15781	cgtctgcaca tcagacttgt cctcagggac tgaggtcact gtcaccttcc ctgtgtctga
  15841	ccacatgacc actgtcccaa gcccccctgc ctgtggtcct gggctcccca gtggggcggt
  15901	cagcttggca gcgtcctggc cgtggactgc ggcatggtgt cctggggttc actgtgtatg
  15961	tgaccctcag aggtggtcac tagttctgag gggatggcct gtccagtcct gacttcctgc
  16021	caagcgctgc tccctggaca cctgtggacg cacagggctg gttcccctga agccccgctt
  16081	gggcagccca gcctctgacc tgctgctcct ggccgcgctc tgctgccccc tgctggctac
  16141	cccatgtgct gcctctagca gagctgtgat ttctcagcat aactgattac tgtctccagt
  16201	actttcatgt ccctgtgacg ggctgagtta gcatttctca cactagagaa ccacagtcct
  16261	cctgtgtaaa gtgatcacac tcctctctgt gggacttttg taaaagattc tgcagccagg
  16321	agtcatgggt ggtcttagct gagaaatgct ggatcagaga gacctgataa ccgatgtgaa
  16381	gaggggaacc tggaagatct tcagttcagt tcatttcagt cattcagttg tgtccgactg
  16441	tttgggatcc catggactgc cacacgccag tcctccctgt ccatcaccaa cttctgaagc
  16501	ttgttcaaac tcatgtccat caagttggag atgcctttca accatctcat cctctgtcat
  16561	ccccttctcc tcccgccttc aatcttccct agcattaggg tcttttccgt gagtcagttc
  16621	ttcgcatcag gtggccaagt tttggagttt cagtttcagc atcagtcctt tcaatgaata
  16681	gtaaggactg atttccttta ggatggactg gtttgatatc cttgcagttc aagggactct
  16741	caagagtctt ctccaacact gcagttaaaa gccatcaatt cttcggtgct cagctttctt
  16801	tttggtacaa ctctcacatt catacatgac taccgaaaat acattagtcg tgtagaacca
  16861	gtttggggct tcccacgtgg ctctagtggt aaagaatatg cctgccaact cagaagatgt
  16921	aagagatgcg gttcaatctc tgggtcggga agatcccctg gagaagggca tgacaaccca
  16981	ctccagtatt tttgcctgga gaatcccatg gacagagaag cctggtggac tgcagtccat
  17041	ggagtctcac agagtcagac acgactgaag caacttagct acttggaaaa gagcatgcac
  17101	gaagctgtct aaaaaacagg tcaagaagtc ttgtgttttg aaggtttact gagaaagttg
  17161	atgcactgct ccaacacttc ctctcagttg aaaagatcag aagcgttaga tcaaatggtg
  17221	gtcaatacct tggatgcgct ccaacaggtt atatctgcag atggaaatga aggcagttta
  17281	tggggtaact ggaggacaag atgagatcat acacttggaa cactgtctgg catcaaaggc
  17341	gtgtacagta aacattagct gttattagca aaataaattc agcttgaatc acccaaatca
  17401	gatggcattc ttaaagccac tgagtggtaa aatcaggggt gtgcagccaa aacgtccatt
  17461	ttgactcatt atgatttcca tgtcacaaga ctagaaagtc actttctcct cagcagaaga
  17521	gaaggtagaa cattttaacc tttttttgga gtgtcaaggg aattttgttt acactgtaaa
  17581	gtcagtgaaa atattgaagc ttttcatttg tggaaaatat taaatatgta aaattgaaat
  17641	tttaaaattt attcctgggt agttttgttt ttccagtagt catgcatgga tgtgagagtt
  17701	ggactataaa gaaagctgag cgctgaagaa ttaatgcttt tgaactgtgg cactggagaa
  17761	gactcttgag agtcccttgg tctgcaagga gatcaaacca gtccatccta aaggaaatca
  17821	gtcctgaata ttcactggaa ggactgatgc tgaagctgaa actccaatac tttggccacc
  17881	tgatgtgaag aactgactca tatgaaaaga ctcagatgct gggaaagatt gaaggtggga
  17941	ggagaagggg acgacagagg atgagatggc tgaatggcat caccgactcg atggacatga
  18001	gtctgaataa gctctgggag ttgttgatgg acagggaggc cctggagtgc tgcagtccat
  18061	gggattgcaa agagttggac atgactgagt gactgaactg aactgagttt ggtaacagat
  18121	atgagaatta tataatttaa atctaaactc ttggtatttc tttctttggc ggttccaaaa
  18181	gagctgtccc ttctgttaac tatataaatc ctttttgaga attactaaat tgataatgtt
  18241	cacaagttat ccaatttctc attactctta gttgtcagta taagaaatcc catttgattt
  18301	atcatgttat agtatctgca actctaatag ttcagttctg acaaattttt attttattta
  18361	aaaatattgg catacagtaa aatttcaaac aatatacaat tctccctttc agtttaaaaa
  18421	acaaaacaaa acaaaagtaa tattagttaa aaaaatccgg gaagaatcca agcatttaaa
  18481	attgcatcac atttctatgc tagacaagct gatataaagt tataattaat aaaggattgg
  18541	actattaaac tctttacata tgaggtaaca tggctctcta gcaaaacatt taaaaatatg
  18601	ttgtgggtaa attattgttg tccttaaaga aataaaaaga cataagcgta agcaattggn
  18661	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  18721	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnna aaatggataa ggggggagga
  18781	catgggtagg ggagcgcgat ggaggaagta aggtggtcga gggagttggg gggggaataa
  18841	gtgggtaaaa gggaagcggg cggaaggagg gggaagcagg agagaggggt gggcgtcaga
  18901	tcggggggag gggtatgagg gagagggaat ggtagacggg gggtgggaag cataaaggaa
  18961	aagatagggg ggggaaaagt tagaagaaga atgaggggat aggcggaaag ggaagagaaa
  19021	tgggagaaga acagaaaaat agggggaggg ggggcgtaaa gagggggggg gagggcaggt
  19081	gtggagatga cagatacggg gaatgccccg gtataaaaga gtatatggcg tggggcgaga
  19141	aggctgtcat cctgtgggag gggggacgcg gagaaccctt cgggctatag ggaggattcg
  19201	gggggatcgt tcgggaaggc agtcagcaca gcacccacca agggtgcagg gatggatctg
  19261	gggtcccaaa gaagaggccc aatcccgcgt cttggcagca aggagccctg gagactggga
  19321	agtgtccagg acactgaccc aggggttcga ggaacccaga agtgtgtctg tgaagatgtg
  19381	ttttgtgggg ggacaggtcc agagctttga gcagaaaagc ggccatggcc tgtggagggc
  19441	caaccacgct gatctttttt aaaaggtttt tgttttgatg tggaccattt ttaaagtctt
  19501	cattgaattt gctacaatat tgtttctggt ttatgctctg gtttcttcgg ctgcaaggtt
  19561	tgtgtgatcg tatctcctca accaggactg aacccacagc ccctgcactg gaaggcgaag
  19621	tcttaaccca gatcgccagg aacgtccctc ccctcactga tctaatccaa gaccctcatt
  19681	aaggaaaaac cgagattcaa agctccccca ggaggactcg gtggggagga gagagccaag
  19741	cactcagcac tcagtccagc acggcgccct ccctgtccag ggcgagggct cggccgaagg
  19801	accaccggag accctgtcgg attcaccagt aggattgtga ggaatttcaa cttacttttt
  19861	aaatctgtct ctcaaggctg ttacaagcgg actttaccag taacttaaaa gttgaaaggg
  19921	acttcccagg cggcacttgc ggtgaagaac ccgccggctg gttttaggag acataagaga
  19981	tgtgggttag atccctggtt caggaggatt cccctggaga aggaaatggc aacccactcc
  20041	agtattcttg cctggaaagc ctcacggaca gaggaggctg gcgggctaca gtccacgggg
  20101	tcgcacacga ctgaatcgac ttagcttcaa gttgagacag gaagaggcag tgactggtgg
  20161	caaaacaccg cacccatgct cccaggggac ctgcagcgct ctggttcatg agctgtgcta
  20221	acaaaaatca acccaacgag aggcccagac agagggaagc tgagttcatc aaacacgggc
  20281	atgatgtgga ggagataatc caggaaggga cctgccaagc ccatgacaga ccggtgtcct
  20341	gtctgagggc cgtcctggca gagcagtgca gggccctccg agaccgcccg agctccagac
  20401	ccggctgggg gctacagggt ggggctgagc tgcaaggact ctgctgtgag ccccacgtca
  20461	gggaggatca ccttgtttgt tttctgagtt tctcttaaaa tagcctttat gggtcctggt
  20521	ctttggtttt aaaataacaa ctgttctccg taaacaacgt gaaaaaaaac aaacaggagg
  20581	aaaacaacgc agcccgggca tttcacccgg aagagccgcc tctaacactt tgacgggttg
  20641	ccttctattt taaccctgtt ttcattgtaa actgtaaaaa ccacatcata aataaattaa
  20701	aggtctctgt gaagtttaaa aagtaagcat ggcggtggcg atggctgtgc cacaccgtga
  20761	acgctcgttt caaaacggta aattctaggg accccctggt ggtccagtgg gtgagatttt
  20821	gcttccattg caggagccgt gggtttgatc cctggttggg gaactaagat cccacatgct
  20881	gtatggagtg gccaaaaaga attttttgta aatggtgagt tttaggtgac gtgaatttcc
  20941	cattgatgca cttcacaggc tcagatgcag ccaggccctc aggaagcccg agtccaccgg
  21001	tcctttactt ttccttagag ttttatggct tctgtttctg cccttaaacc caccatgttt
  21061	caacctcatc tgattttgga ctttataata aagttaggct gtgtttcagg aaactttgct
  21121	cagtattctg taataatcta aatggaaaga atttgaaaaa agagcagaca cttgtacatg
  21181	cataactgaa tcactttggt gtacacctga aactcgagtg cagccgctca gtcgtgtccg
  21241	accctgcgac cccacggact gcagcacgcg ggcttccctg cccatcacca actcccggag
  21301	ttcactcaaa cacatgtccg tcgactcggt gatgccgtcc aaccgtctca tcctctgtcg
  21361	tccccttctc ctcccgcctt caatcttttc cagcatcagg gtcttttcaa atgagtcagt
  21421	tcttcacacc aggtggccag agtattggag tttcagcttc agcatcagcc cttccaacga
  21481	ccccccatac ctgaagctaa cacagtgcta atccactgtg ctgcaacatg aaagaaaaac
  21541	acatttttta agtttaggct gtgtgtgtct tccttctctc aacactgcgt ctgaccccac
  21601	ccacactgcc cagcactgca ttccccgtgg acaggaggcc ccctgcccca cagctgcgtg
  21661	ccggccggtc actgccgagc agacctgccc gcccagagtg gggcccctgg cactggggac
  21721	aaggcagggg cctctccagg gccggtcact gtccactgtt cctactggtt ttgttttcaa
  21781	aagtggaggc agcgtaatat ttccctgatt ataaaaagaa gtacacaggt tctccacaaa
  21841	taaaacaggg gaaaagtata aagaatggaa gttcccagca cagcctggag atcacgccgg
  21901	gtgcacctgg ggtgtccttc caggctggac ctcacatttc acgcagacat cagaaggctg
  21961	cgagatctac ccagaaggct gggtagatgg gggataggtc agtgacaaac agtagacaga
  22021	gagatataca gacagatgat ggatagacag acgctaagac accgagcgag gggacagacg
  22081	gatggaagac accatccttt gtcactgacc acacacccac atgggtgtgg tgagccggct
  22141	gtcatacttg tgaacctgct gctctcacaa caccagctgg gtccctccag ccccagcgtc
  22201	ccacacagca gactcccggc tccatcccca ggcaggaatc ccaccaccaa ctggggtgga
  22261	ccctccccgc aggaaggtcg tgctgtctaa ggccttgaga gcaagttaca gacctacttc
  22321	tgggaagaca gcgcacaacc gcctaccccg cagagcccag gaggacccct gagtcctagg
  22381	gaagggacca cgcggcctgg acggggagcg gccccaggac gctgccccca acctgtccca
  22441	cctcactcct gctctgctct gaggcggggc gcagagaggg gccctgaggc ctcttcccag
  22501	ttcttgggag cacccactgg gcctgaacca ggccagaagc cccctcctca aggtgtcccc
  22561	agaccactcc cctccacctc cggttgctct gtctcctggc agcagggagc cccagtgaga
  22621	agagacagct ccaggctgtg atcttggccc ctggctgctc tggcagtgtg gggggtgggg
  22681	gtcgctggga ggccatgagt gctgggggtc ggggctgtga aagcacctcg aggtcagtgg
  22741	gctgttggtc gggctctgcg aggtccgcac gggtagagct gtgccaggac acaggaggcc
  22801	tggtcagtgg tcccaagagt cagggccaaa ggaaggggtt cgggcccctc tggttcctca
  22861	gcttctgagg ccggggaccc cagtctggcc ttggtagggg ggcgattgga gggtacaacg
  22921	atccaaaaga aaacacacat ctacgaggga agagtcctga ggaggagaga gctacacaga
  22981	gggtctgcac actgcggaca ctgcttggag tctgagagct cgagtgcggg gcacagtgag
  23041	cgaagggagg acggaacctc caaggacacc ggacgccgat ggccagagac acacgcacgt
  23101	cccatgaggg ccggctgctc agacgcaggg gagctcctca ttaaggcctc tcgctgaata
  23161	gtgaggagaa ctggccccgt gtgtggggaa acttagccca gaagaaacgc tgccctggcc
  23221	ccaaggatca nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  23281	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn tgccctttgc
  23341	ctccagggag ggaggaagcg tggatcttgg gtttgccttg ggtttaaagg atccacccac
  23401	tcccttttta gccactccct gtgctggcaa tttcttaaga ctggaggtcg caaagagttg
  23461	gacacactga gcgagtgaac tgcactgagc ctaagaaaag tctttgaatt cctccaaaca
  23521	aaacacactt gtcttgggta ctttccttgg ttttgttaca aatgtctggt ccctctgttc
  23581	tcctggccag ctcctgggtg tcattttgac ctgacgaagt caaagggagc ctggaccctc
  23641	aaaatctgta ggacccagca cccctccatt acacctctgt tcccccgcga acgggcacgt
  23701	gtttcgccgt ctggcgtaat gtgtaagcga cggtgtgata ctcgggagtc ttactctgtt
  23761	tctttttctt ctggggtgac accaccatcc gcacgactct gtctgaatgt gaacatttgg
  23821	gtgatttgat gtggcccaga ctcccccaac gaatgtacct tcaggttggt tttcttcttt
  23881	tatattttgc ttttgtgaat agacacagga tcccatcagt tgtatgtagt gagaaagtaa
  23941	aaacccactc agccttagct ggatggagat ctagtagtaa gatagcacgt tagccggaaa
  24001	tggaaatttc agccagaatc tgaaaagcgt gtcctggaag gagaagaggg actcaggccc
  24061	gagcacactg ctccacgctg gagcctcagg ctctgacagc tgtacctgcc ggggtcttca
  24121	tgggacaggc catgcaggcc acgatcccgt tgagaagttt cttgcctttc catcacattg
  24181	gcaattgcac gctttgctct tgcttctaca tggagtttta cttttatccc agacagtttg
  24241	gtttcttctc tgattttcgc caattgtaca gatcgttaca gtatttctta accacataga
  24301	attcggcagg gggggtgggg ggacagggta gggtggggtg agagtgaggg gagggggctg
  24361	caccgagcag catctggggt cgtagctccc tgacggggat agacctcgtg cccctgcagt
  24421	gacagcacag agtcctcctc tctgaactgc cagggacgct cctgcaattg acttaatgaa
  24481	aggcatctaa ttaggaattt tggggtgaca ttttacattt aagtgtgtga gcagtgatta
  24541	tagttcatat cattttatag tttcgtgatt ttactagctt aaagggtttt tggggtttct
  24601	ttttgtttta aaagctaaaa tctgtttttt aattccatgg aatacaaaaa aaaaaagtct
  24661	gtagaatatt ttaaagagtg aaggctttgt tcggaatgtg agcgctttgc tccactgaac
  24721	cgaacggtaa taacatttgt agaagagacg cagagtgaaa ggtacctctt tttattgagt
  24781	gacatgacag cacccatcgc gtgagttatt ggctggagtt tagagacagg ccatgttggg
  24841	ctaaactcct tattgctgtt ctcagccttt gagtaataat cagaagcttt ctctgaagag
  24901	agtggggtca gctgtcagac tcctaggtgt ctacctgcag cagggctggg attaaatgca
  24961	gcagccagta gatacgggat ggggcaagag gtcaccttgt ccctttgttg ctgctgggag
  25021	agaggcttgt cctggtgcca gtggggccaa agctgtgact ttgtgaccac aggatgtctc
  25081	tgaccctgcc ttgggttccc tgagggtgga gggacagcag ggtctccccg gttccttggc
  25141	cggagaagga ccccccaccc cttgctctct gacatccccc caggacttgc cccggagtag
  25201	gttcttcagg atgggcatcc gggccccacc ctgactcctg gagctggccg gctagagctt
  25261	gctgcagaat gaggccttgg ccattgcggc cctgaaggag ctgcccgtca agctcttccc
  25321	gaggctgttt acggcggcct ttgccaggag gcacacccat gccgtgaagg cgatggtgca
  25381	ggcctggccc ttcccctacc tcccgatggg ggccctgatg aaggactacc agcctcatct
  25441	ggagaccttc caggctgtac ttgatggcct ggacctcctg cttgctgagg aggtccgccg
  25501	taggtaaggt cgacctggca gactggtggg gcctggggtg tgagcaagat gcagccaggc
  25561	caggaagatg aggggtcacc tgggaacagg cgttgggtgt acaggactgg ttgaggctca
  25621	gaggggacaa aaggcacgtg ggcctccccc ccagtgtccc ttaaagtggg aaccaagggg
  25681	gccccggaag ccggaggagc tgtggtgtgt ggagtgcaga gccctcgcgg ggtcctgatg
  25741	cccgtcggac tctgcacagc tcagcgtgtg ccccgcggcc cggtaggcgg tggaagctgc
  25801	aggtgctgga cttgcgccgg aacgcccacc agggacttct ggaccttgtg gtccggcatc
  25861	aaggccagcg tgtgctcact gctggagccc gagtcagccc agcccatgca gaagaggagc
  25921	agggtagagg gttccagggg tgggggctga agcctgtgcc gggccctttg gaggtgctgg
  25981	tcgacctgtg cctcaaggag gacacgctgg acgagaccct ctgctacctg ctgaagaagg
  26041	ccaagcagag gaggagcctg ctgcacctgc gctgccagaa gctgaggatc ttcgccatgc
  26101	ccatgcagag catcaggagg atcctgaggc tggtgcagct ggactccatc caggacctgg
  26161	aggtgaactg cacctggaag ctggctgggc cggatgggca acctgcgcgg ctgctgctgt
  26221	cgtgcatgcg cctgttgccg cgcaccgccc ccgaccggga ggagcactgc gttggccagc
  26281	tcaccgccca gttcctgagc ctgccccacc tgcaggagct ctacctggac tccatctcct
  26341	tcctcaaggg cccgctgcac caggtgctca ggtgaggcgt ggcgccagct ccaaagacca
  26401	gagcaggcct ctcttgtttc gtgcccgctg gggacattgc cagggtgccc ggccactcgg
  26461	aagtcctcac gatgccaccg ctctgaccct gggcatcttg tcaggtcact tccctggtta
  26521	gggtcagagg cgtggcctag gttaaatgct gtcaaagggg actcctttct gggagtccgc
  26581	atagtggggg cttggtgtga tgcccttggg aattctttcc gagagagtga tgtcttagct
  26641	gagataatga cagataacta agcgagaagg acggtccatc aggtgtgagg tttgaagtcc
  26701	aaagctctgt ctctccctcc cacctgcccc ttctgtcctg agctgtttta ggctccaggt
  26761	gagctgtggg aagtgggtga ttctggagat gacaagaagg gatcaggagg ggaaaattgt
  26821	ggctcctaag cagtccagag aagagaaaaa gtcaaataag cattattgtt aaagtggctc
  26881	cagtctcttt aagtccaaat tataattata attttcctct aagacttctg aatacatagg
  26941	aaatcctcag taacaggtta ttgctctgcc ttgaacacag tgataaaagc tgggaggatg
  27001	cagcctaatc tgtctgtgtg aatgagttgt attgattccc tttttggcag ctgcaaactc
  27061	caagcattag gaataaatat gttcactgag aaccccgaag aaagaaagaa agaaaaaaaa
  27121	aaagaattgt aggtgttgat ggacggtttg tggcccctga atatctgggg gatgttcacc
  27181	cagggatcac gtgtaactgc tgggaccccc agccccatgt ccactgcatc cagcctgctg
  27241	ttgaattccg cggatcnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  27301	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnncaat
  27361	tcgagctcgg taccccaaag gtccgtctag tcaaggctat ggtttttcca gtggtcatgt
  27421	atggatgtga gagttggact gtgaagaaag ctgagtgcca aagaattatt cttttgtact
  27481	gggtgttgga gaagactctt gagagtccct tgaactgcaa ggagatccaa ccagtccgtt
  27541	ctaaaggaga tcagtcctga atgttcattg gaaggactga tgctgaagct gaaactccaa
  27601	tactttggcc acctgacgtg aagagttgac tcattggaaa agaccatgat gctgagagga
  27661	attgggggca ggaggagaag gggacgacag aggatgagat ggctggatgg catcaccaac
  27721	tcgatgngac atgagtttgg ttaaactcca ggagttggtg atggacttgg aggcctggtg
  27781	tgctgggatt catggggtcg cagagtcgga catgactgag cgactgaact gaactgaact
  27841	gagctgaaga gctcacctgt accagagctc ctcaggtcct cctgcaggcc tggctgtaat
  27901	ggcccccagg tcaccgtcct gcctccttca tcccatcctt tcacgacagg ctgggagtgg
  27961	ggtgaggtga gttgtcttgt atctagaatt tctgcatgcg accctcagag tgcaatttag
  28021	ctccagagaa ctgagctcca agagttcatt ttttcctttt cttctttatg atactaccct
  28081	cttctgagca gagacctcat gtcagggaga aggggactct gccttcctca gccttttgtt
  28141	cctccaagac ccacacgggg agggtcgcct gcttcactga gccggaaggt tcaattgctc
  28201	atgtcctcca gaaacacccc cccccccaga gacccccaga aataagtgga acagcacctt
  28261	gtttcccaga caagtgggac acacgttatg aaccacctca gtgattaaaa tagtaacctc
  28321	tgtgtatgtg tatttactgg agaaggaaac ggcaacctac tccactattc ctgcctagaa
  28381	aattccatgg gagagaagcc aggcaggcta cagtccacgg ggtcacagag actgaacata
  28441	cacaagcaca tggaagtgta ttttgcagta tttttaaatt tgttcagttc aacatggagt
  28501	acaagaattc aaatcgtgaa gtcaattgac caagaaacca gaagaaatca ctgtgttgtg
  28561	atctctgtgg aggtaacatg ggtacctgtg ctctgaccct cacagcctct ggctctctct
  28621	ctacatgtac atacacatat atttccatgt atgtatgtat tcggaagatt tcacatacgt
  28681	ctcaccagtc cacagccccc gcgttccctg atgcccagaa catctgtgat agctgtgagt
  28741	attgtcacca gataagatct tccaggttcc tgcactcaca ttggttatca ggtctctctg
  28801	atccagcatt tctcagctaa gattccttgt gactcctggc tgcagaatct tctgcaaaag
  28861	tcccacagag aggagtgtga tcactgtaca caggagggcc gtggttctct agtgtgagaa
  28921	aagctaactc agcccgtcac agggacgtga atgtacctga gacagtaatc agttatgctg
  28981	agaaatcaca gctctgctag aggcagcaca tggggtagcc agcagggggc agcagagcac
  29041	ggccaggagc cgcaggtcag aggctgggct gcccaagcgg ggcttcaggg gaaccagccc
  29101	tgcgggtcca caggtgtcca gggagcagcg cttggcagga agtcaggacc ggacaggcca
  29161	tcccctcagg actagtgacc acctctgagg gtcacatcca cagtgaaccc cagagcacca
  29221	tgcctcagtc cacggccagg acgctgccag gctgaccgcc ccactgggga gtccagggga
  29281	gaccacaggc cggggggctt gggacagtga tcatgtggtc agacacagag aaggtgacag
  29341	tgacctcagt ccctgaggac aagtctgatg tgcagacgtg agaagccgag gaggaagctg
  29401	gggacagaca gggctgatgg tgtggtgacc ccgcctctca gtgaggggcc cccgggggtg
  29461	aatttgcata aacccaagcc ctcactgccc ccacaaagct ctgagaggga ataaaggggc
  29521	tcggagagcc cagcactgct gcgggctcag aggcagagct cggggcgcgt ccaccatggc
  29581	ctgggcccct ctcgtactgc ccctcctcac tctctgcgca ggtgcggccc cccagcctcg
  29641	gtccccaagt gaccaggcct caggctggcc tgtcagctca gcacaggggc tgctgcaggg
  29701	aatcggggcc gctgggagga gacgctcttc ccacactccc cttcctctcc tctcttctag
  29761	gtcacctggc ttcttctcag ctgactcagc cgcctgcggt gtccgtgtcc ttgggacaga
  29821	cggccagcat cacctgccag ggagacgact tagaaagcta ttatgctcac tggtaccagc
  29881	agaagccaag ccaggccccc tgtgctggtc atttatgagt ctagtgagag accctcaggg
  29941	atccctgacc ggttctctgg ctccagctca gggaacacgg ccaccctgac catcagcggg
  30001	gcccagactg aggacgaggc cgactattac tgtcagtcat atgacagcag cggtgatcct
  30061	cacagtgaca cagacagacg gggaagtgag acacaaacct tccagtcctg ctcacgctct
  30121	cctccagccc cgggaggact gtgggcacag cagggacagg cctggcccgg ttcccccgga
  30181	gctgagcccc caggcggccc cgcctcccgg ccctccaggc aggctctgca caggggcgtt
  30241	agcagtggac gatgggctgg caggccctgc tgtgtcgggg tctgggctgt ggagtgacct
  30301	ggagaacgga ggcctggatg aggactaaca gagggacaga gactcagtgc taatggcccc
  30361	tgggtgtcca tgtgatgctg gctggaccct cagcagccaa aatctcctgg attgacccca
  30421	gaacttccca gatccagatc cacgtggctt tagaaaggct taggaggtga acaagtgggg
  30481	tgagggctac catggtgacc tggaccagaa ctcctgagac ccatggcacc ccactccagt
  30541	actcttccct ggaaaatccc atggacggag gagcctggaa ggcttcagcc catggggtcg
  30601	ctaagagtca gacacgactg agcgacgtca ctttcccttt tcactttcat gcattggaga
  30661	aggaaatggc aacccagtcc agtgttcctg cctggaaaat cccagggaca ggggagcctg
  30721	gtgggctgcc atccatgggg ccacacagag tcagacacga ctgaagcaac ttagcagcag
  30781	cagcagcagc ccaataaaac tcagcttaag taatggcatc taaatggacc ctattgccaa
  30841	ataaggtcca ctcgcgtgca ctctgtttag gacttcagtt cctgattgtg gagggttccc
  30901	acaagacgtg tgtgtatatt ggtgttgccg gaaaacagtg tcaatgtgag catcccagac
  30961	tcatcaccct cctactccca ctattccatt gtctctgcag gtattaagca taaaggttaa
  31021	gggtcttatt agatggaaga ggagtgaata ctcgtctgtg cttaacacat accaagtacc
  31081	atcaaggtcc ttcctattta ttaacgtgtg ttttaatcag aaatatgcta tgtagaagca
  31141	tccggacgat agcccatgtt acagacgggg aagctgaggc atgaagttct cagcaccttg
  31201	tttcacgtca gacctgaaac ggggcagagc cggcagcaaa caaggttcct cttcccaagc
  31261	gcccgctctt cacccgcttc ctatggcttc tcactgtgct tcctaaacta agctctcccc
  31321	aaccctgtgg agacaggatt agagacttta ggagaaaaga ccaggaacat cccacacccg
  31381	acccgagtga gccactaaga caaggctttg taaggacaga accagcaggt gtcctcagcg
  31441	agccagggag agacctcgca ccaaaaacaa tattgtagca tcctgaccct ggacttctga
  31501	cctccagaaa tgtgaaaaag aaacgtgtgg ggtttaatca actcaccggt gttatttggt
  31561	tatgactgcc tgagttaaga aggagttggg aacacttgag tgtaggtgtt tatggaacat
  31621	aagtcttgtt tctctgaaat aaattcccaa gggtataatt cctaggttgt agggtaactg
  31681	ccacaaatct aggcagctta ttaaaaaaca aagatatcac tttgccagca aaggttcata
  31741	tagtcaaatt atggttttta tagtagtcat gtatggatgt aaaagttgga tcataaagaa
  31801	ggctgagcac cagagaattg atcccttcaa atcgtggtgc tggagaagac tcttgagagt
  31861	cccttggaca gcaaggagat ccaaccagtc aatcctaaag gaaatgaact gtgaatattc
  31921	actggaagga ctgatgctga agctgaagat ccaatacttt ggccacctga tgcgaagagt
  31981	tgactcattg gaaaagaccc tgatgctgga aagcttgagg gcaggaggag aagagggcgg
  32041	cagaggatga gacggttgga tggcatcact gactcaatgg acatgagttt gagccaactc
  32101	tgggagacag tgaaggatag ggaaggctgg cgtggtacag tgcatgcggt cacaaagagt
  32161	ctgacacatc ttagtgactc aacaacgaca gcaacacagg catcacacgc ttagtgtgat
  32221	aagcggcaga actgttttcc aggggtccgn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  32281	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  32341	nnnnnnnnng tacgattcga gctcggaccc tgacattgtg agtcacgtca tgagcagctg
  32401	ttttccggtc ttcagggatt gtggacgatt tctgtttggg tttgctcatg ataatttagt
  32461	tacagcttag gttctttctt tccaggccac gagcgacatg ttttcaggtg agatgacgtg
  32521	gtgggggatg ggcggccaag cccccactgg ggggggaggg attctgttgt gggcaggagt
  32581	tggcagcatc cctgaactga tgacctgcga tccaggtgac aagaaccggg ggatattatt
  32641	cctctgcctt ctcatgtcat gtcctcggtt cttcatgatg aaaacatatg acaatacagg
  32701	ggagttagat ttgggcgggc acaactctgg gtgggggacc cggtggcatt gtgcccagca
  32761	gggccatcaa gatgagggcg acctgggtgg tccccttctc ccctggggtc ttagttttcc
  32821	cctcatggaa atgggatcag gcagcagcca tggaacaccg cgaccgtggc ttctctcacc
  32881	tcctcgtctg tgattttggg tcgggatacc aggcatgaag acctggggcg gggggacatc
  32941	actcctctgc agcagggagg ccgcagagtc ctccgtccat gaggacttcg tccctgggct
  33001	gaccctgcgg actgctggag gctgaagctg gaggcacagg cgggctgcga ggccagggtc
  33061	ctgaggacga cagagccagt ggggctgcag ctctgagcag atggcccctc gccccgggcc
  33121	ctgagcttgt gtgtccagct gcaggttcgc tcaggtgagc cactacgtta tgggggaggc
  33181	gccctgggca gggatcgggg gtgctgactc ctccgagatt ccgaccttct gggagcactc
  33241	tggccacact ctaagcctgg caagagctgg gttcatcagt ctaactctcc tcctgaagtc
  33301	caatggactc tctccatgcg gcagtcactg gatggcctct ttatccccga tggtgtcctt
  33361	ttccgctgac ctggctctcc tgaccacctc ccagcccccc accatacagg aagatggcac
  33421	ctggtccctg cagagctaag tccacccctg gcctggcttc agatgcctac agtcctcctg
  33481	cgggaggccc cgctccccac taggccccaa gcctgccgtg tgagtctcag tctcacctgg
  33541	aaccctcctc atttctcccc agtcctcagc tcccaacccc agaggtatcc cctgcccctt
  33601	tcaaggccct tgtcccttcc tggggggatg gggtgtatgg gagggcaagc ctgatccccc
  33661	gagcctgtgc cgctgacaat gtccgtctct ggatcatcgc tcccctggct ctcagagctc
  33721	cctggtccct ggggatgggt tgcggtgatg acaagtggat ggactctcag gtcacacctg
  33781	tcccttccct aaggaactga cccttaaccc cgacactcgg ccagacccag aaagcacttc
  33841	agacatgtcg gctgataaat gagaaggtct ttattcagga gaaacaggaa cagggaggga
  33901	ggagaggccc ctggtgtgag gcgacctggg taggggctca ggggtccatg gagaggtggg
  33961	ggagggggtg tgggccagag ggcccccgag ggtgggggtc cagggcccta agaacacgct
  34021	gaggtcttca ctgtcttcgt cacggtgctc ccctcgtgcg tgacctcgca gctgtaactg
  34081	cctttcgatt tccagtcgct gcccgtcagg ctcagtagct gctggccgcg tatttgctgt
  34141	tgctctgttt ggaggcccgg gtggtctcca cgttgcgggt gatggtgctg ccgtctgcct
  34201	tccaggccac ggtcacgcta cccgggtaga agtcgctgat gagacacacc agggtggcct
  34261	tgttggcgct gagctcctcg gtggggggcg ggaacagggt gaccgagggt gcggacttgg
  34321	gctgacccgt gtggacagag gagagggtgt aagacgccgg ggaggttctg accttgtccc
  34381	cacggtagcc ctgtttgcct tctctgtgcc ctccgaccct tgccctcagc ccctgggcgg
  34441	cagacagccc ctcagaagcc attgcaatcc actctccaag tgaccagcca aacgtggcct
  34501	cagagtcccc ggctgcgacc agggctgctc tcctccgtcc tcctggcccc gggagtctgt
  34561	gtctgctctt ggcactgacc ccttgagccc tcagcccctg ccagacccct ccgtgacctt
  34621	ccgctcatgc agcccaggtg cctcctccgt gaacccgggt ccccccgccc acctgccagg
  34681	acggtcctga tgggagatgt ggggacaagc gtgctagggt catgtgcgga gccgggcccg
  34741	ggcctccctc tcctcgccca gcccagcctc agctctcctg gccaaagccc ggggctcctc
  34801	tgaggtcctg cctgtctacc gtccgccctg cctgagtgca gggcccctcg cctcacctgc
  34861	cttcagggga cggtgccccc acacagcacc tccaaagacc ccgattctgt gggagtcaga
  34921	gccctgttca tatctcctaa gtccaatgct cgcttcgagg ccagcggagg ccgaccctcg
  34981	gacaggtgtg acccctgggt cccaggggat caggtctccc agactgacga gtttctgccc
  35041	catgggaccc gctcctttct gaccgctgtc ctgagatcct ctggtcagct tgccccgtct
  35101	cagctgtgtc cacccggccc ctcagcccag agcgggcgag acccctctct ctctgccctc
  35161	cagggccttc cctcaggctg ccctctgtgt tcctggggcc tggtcatagc ccccgccgag
  35221	cccccaagct cctgtctggc ctcccggctg gggcatggag ctcacagcac agagcccggg
  35281	gcttggagat gcccctagtc agcaccagcc tctggcccgc accccagcgt ctgccctgca
  35341	agaggggaac aagtccctgc attcctggac caaacaccag ccccggcgcc ccgactggcc
  35401	ccattggacg gtcggccact ggatgctcct gctggttacc ccaagaccaa cccgcctccc
  35461	ctcccggccc cacggagaaa ggtggggatc ggcccttaag gccgggggga cagagaggaa
  35521	gctgccccca gagcaagaga agtgactttc ccgagagagc agagggtgag agaggctggg
  35581	gtagggtgag agccacttac ccaggacggt gacccaggtc ccgccgccta agacaaaata
  35641	cagagactaa gtctcggacc aaaacccgcc gggacagcgc ctggggcctg tcccccgggg
  35701	gggctgggcc gagcgggaac ctgctgggcg tgacgggcgc agggctgcag ccggtggggc
  35761	tgtgtcctcc gctgaggggt gttgtggagc cagccttcca gaggccaggg gaccttgtgt
  35821	cctggaggtg ccctgtgccc agccccctgg ccgaggcagc agccacacac gcccttgggg
  35881	tcacccagtg ccccctcact cggaggctgt cctggccacc actgacgcct tagcgctgag
  35941	ggagacgtgg agcgccgcgt ctgtgcgggg cggcagagga gtaccggcct ggcttggacc
  36001	tgcccagccg ctcctggcct cactgtaagg cctctgggtg ttccttcccc acagtcctca
  36061	cagtccagcc aggcagcttc cttcctgggg ctgtggacac cgggctattc ctcaggcccc
  36121	aagtggggaa ccctgccctt tttctccacc cacggagatg cagttcagtt tgttctcttc
  36181	aatgaacatt ctctgctgtc agatcactgt ctttctgtac atctgtttgt ccatccatcg
  36241	atccaacatc catccatcca tccatcaccc agccatccat ctgtcatcca acatccatcc
  36301	ttccatccat tgtccatcca tctgtccatc ttgcatctgt ctgtccaaca gtggccatca
  36361	agcacccgtc tgccaagccc tgtgtcacac gctgggactt ggtgggggga gccctcgccc
  36421	tcccaccctc ccatctctcc tgaaacttct ggggtcaagt ctaacaaggt cccatcccgt
  36481	ctagtctgag gtccccccgc agcctcctct tccactctct ctgcttctga cccacactgt
  36541	gcactcggac gaccacccag ggcccttgca tccctgtttc cttcctgacc tctttttttt
  36601	ggctctggat ttatacacat tctgcctcct ggaggcgtct cagcttgagt gtcccacaga
  36661	cgcctcagac tcagcatctt ccatcgaaac tgctcccagg tccttgcaga cctggtcccc
  36721	cacattgttc tcaattcggt agatttctcc acaagccaga ggcctggact catcccataa
  36781	tgcctgcccc tcattgagtc agcctctgtg tcctaccata accaaacatc cccttaaaaa
  36841	tctcagaaga acaaaaaaag cacccagatg gcactgtcag agtttatgat gacaagaatc
  36901	ctcagttcag ttcagtcact cagtcgtgtc cgactctttg cgaccccatg aatcgcagca
  36961	cgccaggcct ccctgtccat caccaactcc cggagttcac tcagactcac gtccattgag
  37021	tcagtgatgc catccagcca tctcatcctc tctcgtcccc ttctcctcct gcccccaatc
  37081	cctcccagca tcagagtttt ttccaatgag tcaactcttc gcgtgaggtg accaaagtac
  37141	tggagtttca gcttcagcat cattccttcc aaagaaatcc cagggctgat ctccttcaga
  37201	atggactggt tggatctcct tacagtccaa gggactctca agagtcttct ccaacaccac
  37261	agttcaaaag cctcaattct ttggcgctca gccttcttca cagtccaact ctcacatcca
  37321	tacatgacca caggaaaaac cataaccttg actagatgga cctttgttgg caaagtaatg
  37381	tctctgcttt ttaatatgct atctaggttg ctcataactt tccttccaag aagtaagtgt
  37441	cttttaattt catggctgca atcaacatct gcagtgattt tggagcccca aaaaataaag
  37501	tctgccactg tttccactgt ttccccatct atttcccatg aagtgatggg accagatgcc
  37561	atgatctttg ttttctgaat gttgagcttt aagccaactt ttcactctcc actttcactt
  37621	tcatcaagag gctttttagt tcctcttcac tttctgccat aagggtggtg tcatctgcat
  37681	atctgaggtt attgatattt ctcctggcaa tcttgattcc agtttgtgtt tcttccagtc
  37741	cagtgtttct catgatgtac tctgcatata agttaaataa gcagggtgat aatatacagc
  37801	cttgacgtac tccttttcct atttggaacc agtctgttgt tccatgtcca gttctaactg
  37861	ttgcttcctg acctgcatac agatttctca agaggcaggt caggtggtct ggtattccca
  37921	tctctttcag aattttccac agttgattgt gatccacaca gtcaaaggct ttggcatagt
  37981	caataaagca gaaatagatg tttttctgaa actctcttgc tttttccatg atccagcaga
  38041	tgttggcaat ttgatctctg gttcctctgc cttttctaaa accagcttga acatcaggaa
  38101	gttcacggtt catgtattgc tgaagcctgg cttggagaat tttgagcatt cctttgctag
  38161	cgtgtgagat gagtgcaatt gtgcggcagt ttgagcattc tttggcattg cctttctttg
  38221	ggattggaat gaaaactgac ctgttccagg cctgtggcca ctgttgagtt ttcccaattt
  38281	gctggcatat tgagtgcagc actttcacag catcatcttt caggatttga aatcgctcca
  38341	ctggaattcc atcacctcca ctagctttgt ttgtagtgat gctctctaag gcccacttga
  38401	cttcacattc caggatgtct ggctctagat gagtgatcac accatcgtga ttatctgggt
  38461	cgtgaagatc ttttttgtac agttcttctg tgtattcttg ccacctcttc ttaatatctt
  38521	ctgcttctgt taggcccata ccgtttctgt cctcgcctat cgagccctcg cctccctacg
  38581	tagagactct aagcaggaag gtgacccgtg ctgcactggg tccagcatgc ttttaattca
  38641	gcagtggaac ttctgggtca tgattgtgtt taagggatgc gcatacgatt tttgaagcaa
  38701	aatttaacag gacagcagtg taaagtcagt acttatttct gattaaagaa agcaaatatc
  38761	cagcctgtta ctaagttaat taactaaaga aacatcttca acttaataaa cagtatctcc
  38821	tgaaacttac agcatgcttc acatttaaag gcaaaaccat tttagaggcc agggttccca
  38881	cgcttacgtt tattatttaa tatatgctac agattcaagc ccatgacaca aaatgggggg
  38941	aagagtgtga gtgttaggaa aaatgagata aaattggttt ttgcaggtga tgggctagtt
  39001	tactttaaaa aaaaaaacaa aacaagctca agatgaactg aaggactatt agaactggta
  39061	caagagttaa cctgtgatcg aatacaagca ggctgggcaa aactcagcag gttttcttct
  39121	atacaggcag taatgattga gaatacgaaa cggcggaagc gcttacaacc tcgataacag
  39181	ttctattaaa agccctagga atgaacttaa cacggnnnnn nnnnnnnnnn nnnnnnnnnn
  39241	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  39301	nnnnnnnnnn nnnnngctcc ccccaccctc ccctcctccc cccccaccac cagtgcccca
  39361	ggtctcgtgc ccagagagct gaagatgcca gcaggcccgc tgcctgcctc gctcgcgtgg
  39421	cccgggctcg ctgccggtct gcctgcccag cacacagatg cagccccagc tctcgctgcc
  39481	acccgcctcc cccaggcagg actctcccac aacaccaagg gcgtctctgg gttcaggatg
  39541	gccctcgttg aggtgtaaag tgcttcccgg ggctgagacg aatgggccgg agatccaaac
  39601	gaggccaagg ccgccacggc gcctggcgca gggcacccat ggtgcagagc ggcccagctc
  39661	cctccctccc tccctccctc cctgcttctt tatgctcccg gctatgtcta tttttactct
  39721	gcaatttaga aatgataccg aaggacaaac accgttcccc ctgtgtgtct gctctaaacc
  39781	ctttatctac ttatctatta gcgtgtccaa gttttgctgc taagtgaatg aaggaacact
  39841	acccacaagc agcaacgtcc ccacgaccct cgcctgttca actgggaatg taaatgtgct
  39901	ttcaaaggac ctaagtttct atgttcaaaa ccgttgtgtg tttcttttgg gagtgaacct
  39961	aggccactcg ttgttctgcc tttcaaagca ttcttaacaa ctctccagaa cccagggctt
  40021	ggcttacgtt tccagaaatt ccaaagacag acacttggaa acctgatgaa gaaggcctgt
  40081	gagcacagca ggggccgggg tacctgaggt aggtgggggg ctcggtgctg atggacacgg
  40141	ccttgtactt ctcatcgttg ccgtccagga tctcctccac ctcggaggct ttcagcaggg
  40201	tcacgctggt ggccagggtc gtgtatccat gatctgcaac cagagacggg gctgcggtca
  40261	gcccgcgggc gggcagcagg caggagcagc caggagacgc agcacaccga ggtcctcaca
  40321	tgcaggaggt gggggaagcg gctgtggacc tcacgactgc ccgatgtggg cctcttccaa
  40381	agggccggcc tggaccctgg ctttctccag aggccctgct gggccgtccg cacaggctcc
  40441	agccacaggg cctcttggga caggagggct ccagagtgag ccggccggcg ggaagaggtc
  40501	tgacaccgct gcagtccaca acacgaagcg aggtggagat gggatgaggg atgagaaaca
  40561	cttttctttt aaaacaagag cccagagagt tggaaagagc tgctgcacac gcaacatgaa
  40621	ctcctggccc cggtgccagc ggcgctggga gcccgagttc tcggcaatcc gaccacagct
  40681	tgcctaggga gccgggtgga gacggagggt taggggaagg cggctcccca gggagcgcga
  40741	ggcccggggt cgccaaggct cgccaggggc aagcgcagct aggggcgcag ggttagtgac
  40801	cggcactgca cccggcgcag gagggccagg gaggggctga aaggtcacag cagtgtgtgg
  40861	acaagaggct ccggctcctg cgttaaaaga acgcggtgga cagaccacga cagcgccacg
  40921	gacacactca taccggacgg actgcggagt gcacgcgcgc gcacacacac acacacacca
  40981	cacacacaca cacacggccc gggacacact cataccggac ggactgcgga gtgcacgcgc
  41041	acacacacac ccaccacaca cacacccacc acacacacac ccaccacaca cacacacaca
  41101	cacacacacc cccacacaca cccacacaca cccacacaca cccacacaca cacacccaca
  41161	cacacacaca cacacacaca cacacacacg gcccggtggc cccaggcgca cacagcacgg
  41221	agcaaacatg cacagagcac agagcgagcg ctagcggacc ggctgccaga ccaggcgcca
  41281	cgcgatggat tgggggcggg gacggggagg ggcgggagca aacggnnnnn nnnnnnnnnn
  41341	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  41401	nnnnnnnnnn nnnnnnnnnn nnnnngtatt aaagaagccg ggagcgagaa tatgacggca
  41461	agaggatgta ggtgggggcg gggcaagagt aaagagagcg gacggtagag gggatgcgat
  41521	tgtgatgcgg aagcgagacg aggagtgatg ccgtattaga ttgatagcaa gaggaacagt
  41581	aggagggggg ggggagagga gggggaggtg gggggtggtg ggtgggaagg gaactttaaa
  41641	aaaaagaggg gagagttgga ggggggaata aacgggcggt aaaaaagaac aatttgaaat
  41701	taccagggtg gggcggccag gggggtgatt cattcttgga gggggcaaca tatggggggt
  41761	ggctgtcgcg gattaggaga aaataaatat caggggtgat taagtgtttg gcgttgggga
  41821	ataatgaagt aagaatcaaa tatgaatcgc gttggcatcg ttagccatcg ggggaaacat
  41881	ttcccatgca aggaacaagg atgtgagaat gcgtccgtct gaaccaccgt cccggggtcc
  41941	cagtaggact cgccgagctg atagttgccg gagcaacagt taagggagca gaagctgcta
  42001	caaaaccacc acctgccaaa gtagggtctc caattacgga gtgcgcctcc tgggtgtcgg
  42061	tccaaacctt tggaaaggac ctggaaataa gtgctaccca ccagatatta atataaaccc
  42121	acctggccag gagaggcagg cgctgctggc acaggaagtg tccccagact cagtcatcaa
  42181	ggtaaataat attttgggac ctccctggaa atccagtggt taggactctg cggttcaatc
  42241	cctggtcggg gaactaagat cccacaagtc acaagacatg gccaaattta aaaaagaaaa
  42301	aaagagagag aaatatttag tgcaataggt tttagaattg aaattaagct cctgcccacc
  42361	cccacccccc aatctggatg aataaagcat tgaaatagta agtgaagtca ggctctgaca
  42421	tgcactgatg tgactcacct taagcaaccc ccaccctagg actggtcggg gttccaggag
  42481	tttcaggggt gccaggaaga tggagtccag cccctgccct ctccccccac cacgtcctcc
  42541	actggagccg cctaccccac ctcccacccc tccgcaccct gctacccccc acccctgccc
  42601	ccaggtctcc cctgtcctgt gtctgagctc cacactttct gggcagtgtc tccctctaca
  42661	gctggtttct gctgcccgct accgggcccg tcccctctgt tcagttcagt tcagtcgctc
  42721	agtcatgtct gactctttgt gaccccatgg actgcagcac accaggcctc cctggccatc
  42781	accaaccccc agaacttact caaactcatg tccatcgagc cagtgatgcc atccaaccat
  42841	ctcatcctct gtcgacccct tctcctggcc tcaatctttc ccagcatcag ggtcttttcc
  42901	aatgagtcag ttctttgcat caggtagcca aagtattgga gtttcagctt cagcatcatt
  42961	tcttccaatg aatattcagg actcatttcc tttgggatga actggttgga tctccttgca
  43021	gtccaaggga ctctcaagag tcttctccaa caccacagtt caaaagcatc aattcttcag
  43081	tgctcagctc tctttatagt ccaactctca catccatacg tgaccactgg aaaaaccata
  43141	gcctcgacta gatggaactt tgtgggcaaa gtaatgtctc tgcttttgaa tatgctgtct
  43201	aggttggtca taacttttct tccaaggagc aagcgtcttt taatttcatg gctgcagtca
  43261	ccatctgcag tgatttttgg agcccaagaa aataaagtct gtcactgttt ccactgtttc
  43321	cccgtctatt taacggaggg aaatttccca gagcccccag gttccaggct gggccccacc
  43381	ccactcccat gtcccagaga gcctggtcct cccaggctcc cggctggcgc tggtaagtcc
  43441	caggatatag tctttacatc aagttgctgt gtgtcttagg aaagaaactc tccctctctg
  43501	tgcctctgtt ccctcatccg cagaagtgac tgccaggtcg gggagtctgt gacgtctcca
  43561	gaagccggag gattttctcc ccatttgctg aaagagagct cggggtgggg gaagcttctg
  43621	cacccctagg atcaccagag gagccagggt cttcagggtt cccggggacc cctcagtggg
  43681	ggctcaggaa ccacagagcc agaccctgat tccaaaaacc tggtcacacc tccagatgac
  43741	cctttgtccc ttggctccgc ctcaaatgct ccaagcccca acagtgaagc gcttaagaga
  43801	aggatccacc aggcttgagt ttggggagga gggaagtggg gagctggggg agggcctggg
  43861	cctgggagac aggaatccac catggcttca ggcagggtct ctggggcctg cggggtggag
  43921	agcgggcagg agcagacaga ggtgactgga cacgacacac ccctccactc caagggaggt
  43981	gggcaggggc ggggcacaga ggaacaagag accctgagaa ggggtccacc gagcagactg
  44041	ctggacccag acatctctga gccagctgga atccagctct aagccatgct cagcccaggc
  44101	agggtatagg gcaggactga gtggagtggc cagagctgca gctgcatggg ctgggaaggc
  44161	cctgcccgtc ccctgagggt cccccagggt ctagccagac tccaatttcc gaccgcagca
  44221	cacacaggag gaagtggtcg gggtggagtt ggcccagagg tctgggcagg tgcagggtgg
  44281	gggaaggggg gcagctggag tcacccgctg aattcaggga cagtcccttt ttctccctga
  44341	aacctggggc tgtcccgggg gccaccgcag cctccaggca gcggggggac ccagccccca
  44401	atatgtgaga agagcaggtc ccaggctgga gagagcgaag caccatggtg gggagaagtt
  44461	agactggatc ggggccccta ggggctcccc cggacctgca cggcagccgt cagggcaccc
  44521	gcaccccatt gctgttcagt gctggccagt gtccaaggcc agggatgtgt gtgtgtgtgt
  44581	gtgcgtgcgt gcgtgcgtgt gtgtgtgcgt gtgtgcgcgt gcgtgcgtgt gtgtgtgtgt
  44641	gcgtgcgtgt gcgtgcgtag acgtgtgcgt gcgtgcgtgc gtgcgtgcgt gtgtgtgcgc
  44701	acgcgcgcag cccagcctca gcactggacc aggcagcctg ggattcctcc aaaactgcct
  44761	tgtgagtttg gtcaaaccgt gaggctctga tcaccgccat ccattcgccc cctcctgccc
  44821	ccctcatcac cgtggttgtt gtcattatcg agagctgtgg agggtctggg aggtcatccc
  44881	acctgccagc taaaccgtga ggctgccgca atcgcactga tgcgggcaga cccgagacgc
  44941	tgtgccggag acgaaggcca gcttgtcacc ccgccagagc ggcagtcggg ccacaagcat
  45001	catccaagca gtggttctct gagcccgacg gggtgatgca aaggagccag gagacacctg
  45061	cgcgtccaag ctgggggacc ccaggtctgt tatgccggac agtaaacacg ttcagctccg
  45121	gagggagagg gttcccctac cttccagggt ttctcattcc acaaacatcc aaagacaatc
  45181	cataccgaag gcgatccgtg cctttgctcc tgagacgtgc ggaagcacag agatccacag
  45241	acactgtctc ccaggatcct atgtatgtaa aggaaccgaa gtcccaggct gtgtgtctgg
  45301	taccacatcc cacggaacag gctggactga ttttcaccaa atgtagcaga aacgttaagg
  45361	agtatcagct tcaaaatatg agggccagac atgtctgaga agtcccttcc agaaaagtcc
  45421	ctttggggtc cttccccaga gttgctgaaa cagagaaccg gaagggctgc agagctgaac
  45481	ttaaacaact ggatcgcaaa ggtccgtctc atcagagcga tggtttttcc agtggtcatg
  45541	tatggatgag agagttggac cataaagaaa gctgagcgcc gaagaatcga tgcttttgaa
  45601	ctctggtgtt ggagaagact cttgagagtc ccttggactg caaggagatc caaccagtca
  45661	atcctaaagg aaatcaatcc tgaatattca tgggaaggac tgatgctgaa gctgaaactc
  45721	caatactttg gccacttgat gcaaagaact gactcactgg aaaaaccctg atgctgggaa
  45781	aggttgaagg caggaggaga aggggtcgac agaggatgag atggttgggt ggcatcaccc
  45841	acccatggac tcaatggaca tgggtttgag taaactctgg gagttggtga tggacagaga
  45901	atcctggcat gctgcggtcc atggggtcat agagagtcag acacaactga gcgactgaca
  45961	gaactgaagc aactggcaag ccggagggta ggtgccggct gcgatgagcg ggaacgtgca
  46021	acctgccacg tggagctctt cctacaccca gagtcctgac ggcactggga ccctagccct
  46081	ccacggcctc tccagggcca cgagacaccc tcacagagca gagaagcgga acagagctgg
  46141	tgtgcagaac caggccccgg gggtggggcg gggctggtgg gcaggcttta gtgagaagcc
  46201	cttgagccct ggaaccagag cagagcagaa cagttggcag aggcccccct gggagaggcc
  46261	ccccgcccag agtaccggcc ctgggccctg ggggagaggg cggtgctggg ggcagggaca
  46321	gaaggcccag gcagaggatg ggccccgtgg gacggggcgc accaaaacag cccctgccag
  46381	caaggggaag ctggggcact ttcgaccccc tccaaggagg agcccacacc agcgcatctg
  46441	cccaaggtgc ccttggccct gggggcacat gaggcccagg ccaggccagg gggcccatga
  46501	ggcccccagg ggtcagtgca gtgtccccag gcagccctgg cctctcatcc tgctgggcct
  46561	ggcctcttat cccgtgggcg cccacggcct gctgcccccg acagcggcgc ctcagagcac
  46621	agccccccgc atggaagccc cgtcaggaaa gagcccttgg agcctgcagg acaggtaagg
  46681	gccgagggag tcatggtgca gggaagtggg gcttcccttc gatgggaccc aggggtgaat
  46741	gaccgcaggg gcggggaacg agaagggaaa ccagctggag agaaggagcc tgggcagacg
  46801	tggctgcacg cacagcgctg accctgggcc cagtgtgcct ttgtgttggg ttttattttt
  46861	aattttgtat tgagatgcta tttatctcgt ggagcttttg ccgccctgag attttgtacc
  46921	cgtggctggt gtccctcttg cctcaccccg gcctctgtag cagggcagac acggcgcaac
  46981	ggggcagggc gtgcccagga ggcactgtca ttttgggggc agcggcccca caaggcaggt
  47041	ctgccttcct cccctcttac aggcagcgac agaggtccag agaggtgagg caagctgccc
  47101	aatgtcacac agcacacggg cgcagtccca ggactgtaga aatcccggga ctagacaggc
  47161	accagagtgt cctgtgtttt taaaaaaacg gcccaagaga agaggcaagt ctgcaaggcg
  47221	tcccgggaag gcagcagggg cttggctcgg tctcccccaa ggaggccagc tcctcagcga
  47281	ggttcctaag tgtctaacgg agccaagcct gaaccaaggg ggtcacgtgc agctatggga
  47341	cactgacctg ggatggggga gctccaggca aagggagtag ggaggccaag gaggagagag
  47401	gggtgcacag gcctgcaggg agcttccaga gctggggaaa acggggttca gaccacgggg
  47461	tcatgtccac ccctccttta tcctgggatc cggggcaggt attgagggat ttatgtgcgg
  47521	ggctgtcagg gtccagttcg tgctgtggaa aaattgtttc agatcagaga ccagcgtgag
  47581	gtcaggttag aggatggaga agaagctgtg aaaaggtgat ggagagcggg gggacggtcc
  47641	tcggtgatca ggcaccgaga tcgcccatgg aatccgcagg cgaatttaca gtgacgtcgt
  47701	cagagggctg tcggggagga acaggcactg tcatgaactg gctacaaaaa tctaaaatgt
  47761	gcaccctttt cggcaatatg cagcaagtca taaaagaaaa cgcatttctt taaaattgcg
  47821	taattccgct tttaggaatt catctggggg cgggggaaca atcaaaaaga tgtgaccaaa
  47881	ggtttacaag ccaggaagtc aactcgttaa tgatgggaga aaaccggaaa taacctgaat
  47941	atccaacaga aagggtgtga tgaagcgcag catggcacat ccaccgcaag gaatcctaac
  48001	acaaacttcc aaaacaatat ttctgacgtt gggtttttaa agcatgcgtg cactttcaaa
  48061	agcttgtcag aaaacataga aatatgccaa taatgtgtct ctagccaaat tttttaattt
  48121	ttgctttata attttataaa gttataattg tatgaaatat aatgataaaa ttataaacta
  48181	taaaaaagtt atgaaaatgt tcacaagaag atatacatgt aattttatct tctacaatac
  48241	tttttaatac cagaataacg tgcttttaaa aaagattgag cacagaagcg tataaagtaa
  48301	aaattgagag tttctgctca ccaaccacac gtcttacctt aaaacccatt ctccagcgag
  48361	agacagtgtc atgtgggtct gtacacttct ggcctttctc ctaggcatgt atgtccctga
  48421	aaactcacac acacggctaa tggtgctggg attttagttt tcaaaacgga ctcatactct
  48481	gcctatgagc ctgcaactat ttattcagtc tgttgagatt ttctatatca gcccacatgg
  48541	atcccgcatg ttctctgaat ggctctgtat gaattcaaag tttggaagaa gcagcgtgtc
  48601	tttaatcatt cgcctattaa tggacgtttg gggtgtttcc actacaaaan nnnnnnnnnn
  48661	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  48721	nnnnnnnnnn nnnnnnnnnn nnnnnnnnng atacaattcg agctcggtac cctggcttga
  48781	actatatgaa cagagaacga tgagaacagt ttctcaaact tggaacagtt aacattttgg
  48841	gctaaatgat tcttttttgt gtggagttgg cctatgaata gaggatatta gcagcatcat
  48901	ttaaccttta ctcactacat acctgtagca actacatcct ctccatttgt gtcaatcaaa
  48961	actgtctccg gacatggaca agtgtgcccc tgggatgggt ggaatgacct tttgttaaga
  49021	accactgggt cagagattca tagatttttg tcttgttgac tttttaaaaa tacatcttgg
  49081	tttttatttt attggtttct gctcttatct ttatgattac cttcctttta cttggggctt
  49141	ccctgataga ttttcccttc tggctcagct ggtaaagaat ctgcctgcaa tgcaggagac
  49201	ctgggttcag tccctgggtt gggaggatcc cctggagagg agaagggcta cccaccccag
  49261	tattctggcc tggaggattc catggagtgt atagtccatg gggtcgcaga gtcggacatg
  49321	actgagtgac tttcacacac acatatgtcc ctggtagctc agctagtaaa gaatcccacc
  49381	cgcaatgcag gagaccccgg tccaattcct gggtccggaa gattcccttt tgtttactcc
  49441	ataagatctt atctggggac aaaactaaca gctatgccag accttctgga catcagggaa
  49501	cgtgaggggt gtggactgga cagatgtgtg tgttctccca aacacaaaca tacatctgta
  49561	tacatgtaca tggagagagg gggagggagg ctgtgagtct ccaggggacc gtgcaaccat
  49621	gtgacattca tggaggcgtt tgcgggtgat cactacacag tttcttcttc tggtttcttg
  49681	gtcaattgac ttcacaattc caattcctat acttcatttt agactgaggg aattttacac
  49741	tattgtaaga catatgtata catgagttat gttcagcgcc atgagggctc attttgtgtg
  49801	tccactttgc ctggaaacaa agttggactg atttacttct aggggtgcct gggggtgttt
  49861	ctggaggaca ggagcatttg aacccaaggg ctcggtgaag catgagcctc tctgcaggtg
  49921	gacccaggag gaacgcaagg ccgaggaagg cagactctcc tcctccctaa cccgaggtct
  49981	ctgctcagaa aagggacaat ataatgacta gaagaaaaga aagaacatca gctgtgggag
  50041	gtttgttctc tggagcagat tcacacgttg aggctcatgt gcaggaattc taggtgaaac
  50101	agagcagtca cccatgtgtg ttggaaaatt ttaaattaca tttgcagtta cgactttgtt
  50161	taagccagac agggtagcac agcaaagtca ccatgtggtc acctgtgttt tgtaaaggag
  50221	agagaacttg ctggcacatt caggaaaggc cgtgtctcag ctttggaggc acactgagag
  50281	gccacaagca gatggtgagg accagggtct cgggcagagg gatcaattca ctgctcttca
  50341	cttttgccac atctgtgtgc tgtccatcct ggccagagta gttcagtctt cagatgctgg
  50401	agttcccatt ggtagaaatc caatctgggt catttttaaa cctctcttgg ttctacttaa
  50461	tggttttaaa atctctttgg ctcaagaaaa aaaataaaca taattttaaa gggtggtttg
  50521	gggccttgac tataaagtac attatctggg ccatttcaga gcatggttga attaatacat
  50581	ttcgtgctta ctatagctcc tattttcttg attctttaca ggtaattttt gttaggaatc
  50641	gggtactgtg aatattttct tgttgaatac gggatctttg tattttttcc taattttttt
  50701	ttttttttca tttttggttt taccttcagg aaagtcacta ggactcagga aagtcctttg
  50761	tccgcctgtt atttcagtct cttacctggg gccagggcag cgtttcctct gggctaagtt
  50821	tccccacaac cggggccagt tctcctcact cttcaccctg aggccttaat gaggagctcc
  50881	cctgcgtctg agcagccggc cctcctgtga cgtgcgtgtg tctctggcca tcggcgtccg
  50941	gtgtccttgg aggttccgtc ctcccttcgc tcactgtgcc ccgcactcga gctctcaggc
  51001	tccaagcagt gtccgcagtg tgcagaccct ctgtgtagct ctctcctcct caggactctt
  51061	ccctctagat gtgtgttttc ttttggctcc ttggacctcc gctctgaacg caggcctggt
  51121	gctgagtgtg atctctggag ggaagcctgg gaggctggac gggtccgccc tgcggtgtgg
  51181	tgacaggtgt gggctcgggg cggggcctgc acgtcgtcct gacccgagcc gggactgggc
  51241	tccgggcctc aggcatcact gactgaatct ccctcacaga ggggtcaggg cctgggcggg
  51301	ggaaccgtct ctgcaatgac agcccctccc agggagggca cagcggggag ctgccgaggc
  51361	tccagcccta gtgggaggtc ggggagccca ggggagcggc ctgacggccc cacaccggcc
  51421	cagggctggt tcgttctgtt tctcgagctc aacagaagct ccgaggagct gggcagttct
  51481	ctgaattcgt cccggagttt tggctgctga gtgtcctgtc agcaccgtat ggacatccag
  51541	agtccattag cagtggtctc tgtccctctg tctgtccttc atcaggctct ttgtccaggt
  51601	caccacacgg ccaacaccag gacagtctgg tcccgccagc ccatcgtccc tgcggacgcc
  51661	cctgtgcagc ctgccgaagg gccgggaggc cgggggaacc gggccaggcc tgtccctgct
  51721	gtgtccacag tcctcccggg gctggaggag agcgtgagca ggacgggagg gtttgtgtct
  51781	cacttccccg tctgtctgtg tcactgtgag gattatcact gctgtcagct gactgacagt
  51841	aatagtcggc ctcgtcctcg gtctgggccc cgctgatggt cagcgtggct gttttgcctg
  51901	agctggagcc agagaaccgg tcagagatcc ctgagggccg ctcactatct ttataaatga
  51961	ccctcacagg gccctggccc ggcttctgct ggtaccactg agtatattgt tcatccagca
  52021	ggtcccccga gcaggtgatc ttggccgtct gtcccaaggc cactgacact gaagtcggct
  52081	gggtcagttc ataggagacc acggagccgg aagagaggag ggagagggga tgagaaagaa
  52141	ggaccccttc cccgggcatc ccaccctgag gcggtgcctg gagtgcactc tgggttcggg
  52201	gcaggcccca gcccagggtc ctgtgtggcc ggagcctgcg ggcagggccg gggggccgca
  52261	cctgtgcaga gagtgaggag gggcagcagg agaggggtcc aggccatggt ggatgcgccc
  52321	cgagctctgc ctctgagccc gcagcagcac tgggctctct gagacccttt attccctctc
  52381	agagctttgc aggggccagt gagggtttgg gtttatgcaa attcaccccc gggggcccct
  52441	cactgagagg cggggtcacc acaccatcag ccctgtctgt ccccagcttc ctcctcggct
  52501	tctcacgtct gcacatcaga cttgtcctca gggactgagg tcactgtcac cttccccgtc
  52561	tctgaccaca tgaccactgt cccaagcccc ccggcctgtg gtctcccctg gactccccag
  52621	tggggcggtc agcctggcag catcctggcc gtggactgag gcatggtgct ctggggttca
  52681	ctgtggatgt gaccctcaga ggtggtcact agtcctgagg ggatggcctg tccagtcctg
  52741	acttcctgcc aagcgctgct ccttggacag ctgtggaccc gcagggctgc ttcccctgaa
  52801	gctccccttg ggcagcccag cctctgacct gctgctcctg gccacgctct gctgccccct
  52861	gctggtggag gacgatcagg gcagcggctc ccctcccgca ggtcacccca aggcccctgt
  52921	cagcagagag ggtgtggacc tgggagtcca gccctgcctg gcccagcact agaggccgcc
  52981	tgcaccggga agttgctgtg ctgtgaccct gtctcagggc ggagatgacc gcgccgtccc
  53041	tttggtttgt tagtggagtg gagggtccgg gatgactcta gccgtaaact gccaggctcc
  53101	gtagcaacct gtgcgatgcc cccggggacc cagggctcct tgtgctggtg taccaaggtt
  53161	ggcactagtc ccaccccagg agggcacttc gctgatggtg ttcctggcag ttgagtgcat
  53221	ttgagaactt acatcatttt catcatcaca tcttcatcac cagtatcatc accaccatca
  53281	ccattccatc atctcttctc tctttttctt ttatgtcatc tcacaatctc acacccctca
  53341	agagtttgca ttggtagcat atttacttta gcacagtgtg cctcttttta ggaaactggg
  53401	ggtctcctgc tgatacccct gggaacccat ccagaaattg tactgatggc tgaacccctg
  53461	cgtttggatt cttgccgagg agaccctagg gcctcaaagt tctctgaatc actcccatag
  53521	ttaacaacac tcattgggcc tttttatact ttaatttgga aaaatatcct tgaagttagt
  53581	acctacctcc acattttaca gcaggtaaag ctgcttcgca tttgagagca agtccccaga
  53641	tcaataaaga gaatgggatg aacccaggat ggggcccagg ggtcctggat tcagactcca
  53701	gccgtttagg acagaacttg actaggtacg aagtgagcgg ggtggggggg caatctgggg
  53761	ggaactgtgg cacccccagg gctcggggcc atccccacca catcctggct ttcatcagta
  53821	gccccctcag cctgcgtgtg gaggaggcca gggaagctat ggtccaggtc atgctggaga
  53881	atatgtgggg ctggggtgct gctgggtcct aggggtctgg ccaggtcctg ctgcctctgc
  53941	tgggcagtga taattggtcc tcatcctcct gagaagtcac gagtgacagg tgtctcatgg
  54001	ccaagctatt ggaggaggca gtgagcactc ccacccctgc agacatctct ggaggcatca
  54061	gtggtcctgt aggtggtcct ggggcttggg ccgggggacc tgagattcag ccattgactc
  54121	tcagaggggc cagctgtggg tgcagcggca gggctgggcg gtggaggata cctcaccaga
  54181	gccaaaataa gagatcaccc aacggataga aattgactca caccctttgg tctggcacat
  54241	tctgtcttga aatttcttgt ggacaggaca cagtccctgg ataaagggat ttctatcttg
  54301	cgtgtgcaat agagctgtcg acacgcttgg ctgggacatg taatcctttg aacatggtat
  54361	taaattctgt tcactaacat ctgaaaggat ttttgcatca ataaacctaa ggtatattgc
  54421	cctgtcattt ccttgtcttg tagtgtctct gagtaggctg gaaggggtaa ccagcttcac
  54481	aaatcgagtt aggaaattcc cttattcttc cactgtctaa tagactttca taagattagt
  54541	gttaattcct ctttaaatcg ctgctataat catcactgtg gccaccggta ctgaattttt
  54601	tgttaggatg atttttaaac aagcatttta atgatttttc cttttatttt cggctgtgct
  54661	gggtctcgtt gctgtgtgcc ggcgttctct cgctgtggcc agtgggggcg ctgctctcgc
  54721	gttgcgaagc tcgggcttct gactgcagtg gcttctctcg ttgcagagcg cgggctccag
  54781	ggcgctcagg ctcgcgtggc tgcggcacgt gggctcagta gtcctggggc acaggtgcag
  54841	cagcctctca ggacgttttg ttcccagatg gtgggtcggt cgaaccggtg tcccctgcgt
  54901	tgcaaggtgg attcttcacc gctggaccac cagcgacgtt ccctggaggt ttttaattat
  54961	ggatttaagc tctcattaga tgtctcctca catttcctat ttctttttga gtcagtttga
  55021	tactttgttt gtgtctgtaa gtttgtccat tttatccaag tcatctaatg tgttgataga
  55081	caattattgg ttagtcatct aattgttggt ttacaatttt gagagcattg tcctgcaatt
  55141	ccttctatct gcaagattgg taataatatc tcccaagagg agtcacaaac tgaaatgaga
  55201	ttanatacag gctttttttt taaaagaatg aacttatgtt gttgcctttc tcatagatct
  55261	tacttcttag catgactgta cttactgact ggggcgtttt catgtctgtg tggagagcta
  55321	ccattagtac ttcttatcgc ccaaagacat cgggctcctg ggcacagtga aaacactcct
  55381	ttctgtggct attttgcaaa atatggccta gcctagcgtc ataagggatc acagctgaca
  55441	actgctggaa cagagggaca tgcgaagcaa cgtgagggct ggaacctgga gggtcctctc
  55501	tggggacagt ttaaccagct ataatggaca ttccagcatc tgggacatgg agctgtgaac
  55561	tggaccaatg actgtcattt ttggaagaga aatcccagga gagaagggtc caggggaatc
  55621	tgaggccgca tgcagtgcct caggacaggg gacaccttct ccagcagagc aggggggccc
  55681	gcccaggccg cctgcagtga ttccaccagg aggagatgca tccctgcaga cctctgacag
  55741	cacggccctc tcctgagaca cagggtcaca cccggggccc tggaaccctt tgagacccta
  55801	aacctttcct ttcctgacca ccctgacagc agtctagctc agaacagaca tcttcatttt
  55861	cagcaggaaa atccttttcc tcgtttgagg gagcgactgg caccggagga gctgagtctt
  55921	ttaaacacag gctgcctgaa cctcagggat gacctgcagc tgctcagagg aggctggagt
  55981	gtgatagctc actctaatgt tactaaaagg aacatattgg acaccccctc tctgaaaaat
  56041	ttccctcctg cctctcatct cttagtccac tttatcgccg ttttactgct tttctattta
  56101	ctactcttaa cgccaaccta tcttatttcc cctcccagtt taacacggtt ttccctccac
  56161	ccgctctctt taatctcaga agattctgcc tattcctcta ttatcacacg cccctacttt
  56221	ttattttttt tcttacccgc cttttattcc ctcccctcct cactctctat ttaattacat
  56281	cttaactaca ccgcctgcgc tatcttcgaa tgtatccaaa tatttttccc ttatataaca
  56341	ctccaggccg agcggctaac ttattataat ttctttatag cgcctaccta atttcccttt
  56401	atttctaatt atctatatat acccatgcaa tttcgnnnnn nnnnnnnnnn nnnnnnnnnn
  56461	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  56521	nnnnnnnnnn nnnnntgggt gtacgttata gagtaaacgc gcatgaagaa gtgggtcaat
  56581	ctatggctgt gagaggcaga aaataatatt atcatatata atttatgtta taacacactg
  56641	aggtggtggg ctcgtagaat agtgcggacg gggagaaagg tgggaaggag aagacacaag
  56701	agagagatgt tcgcctcgcg ggatggatgg gcggagggat agaagaataa aaagaggaga
  56761	ggtatagagg ggggcggggg gcataacgtg tggtggggta aatagtaggc ggtaattatg
  56821	aaaaaaagaa agacgggggg ggcggtaaca tagaatacgc aaaaaagtca tatactgaac
  56881	ggggattagg gagaagaggt ggggggcgtg gggtgcgggg gaaagaggtg tgtgtataat
  56941	tggtatggag tgttatttga atatatatta atgtaatagg gagtgtaatt agtgaaattg
  57001	tgggagtatt atattggggt gtgggggaca tggcaaagtg atgatcggga taaaaaaagt
  57061	aaagcaagag gggaggggaa aataaggggg gggagaaggt cgaagaaaat aagaggaaga
  57121	agaaagaacg ggggtggcgg gcgggggggg cgccgctctt gtatctggct tttttgttgt
  57181	gtcggtggtt gttcgcgtct tgttgggtcc ggggcgggtg tgcggaaaaa aaaaaaggcg
  57241	ggaggcccgg ggcccggtca cgcggcaccc ccgcgggtcc ctggcttctc cttcggcagc
  57301	tccgggggtc ggtgagcctg cgccctccgg gccgccggcc cgagctgtgt gcgccctgga
  57361	gaatcggagc cgctgtggca gcacgcggag ggcgcgcgca agggccacgg gacggacctt
  57421	caaaggccgc ggcggagcgc ggcaagccga accgagggcg gtctggcgat cggccgagcc
  57481	ctgctccccc ctcccgcgtg gccccagggt cgcgggtgga ctggggcggg tacaaagcac
  57541	tcacccccgt cccgccccca gaaagcctcc caggactctc acagagcacc cgccaggagg
  57601	catccggttc ccccctcggc tcagttcagt tgctcagtcg tgtccaactc tttgcgaccc
  57661	catggactgc agcaccccaa gcttccctgt ccatcaccaa ctcccggagt ttactcaaac
  57721	tcatctattg agtcagtgat gccatccaac cgtctcatcc tctgttgtcc ccttctcctc
  57781	ccactttcaa tctttcccag catcagggtc ttttcttatg agccagttct tcacatcagg
  57841	tggtcagagt attggagttt cagcttcagc atcagtcctt ccaatgaaca ctcaggactg
  57901	atttccttta ggatggactg gctggatgca gcgccagaca ccgaccgcgt ttaccccgtg
  57961	tgtcctttcc aatggctgtc ccctgcgggc ctaggggcat tggtgcgggt ttgaatcctg
  58021	tggccttgaa ttttacgcct tagttccagg tccagggcag ggccatccgg attcaggatg
  58081	cttcccagcc cttcaggaat ggcaggtttt catggtcctt tctgagtgag ttctgagtgg
  58141	tcatattggt gcccttggca gggagggctc ctgactttcc tatcttcaca tcactgtccc
  58201	caacccccaa gagaggcctc ttggcccagg gactgcaggg aggatgaagt caggagcaga
  58261	agcatggggt agggggctca ggtgggcaga ggaggcccct ctgtgaggag gaacggcaag
  58321	cgaggaggga acaggggcac cggcagtgcc tggcaagctg ggtgatgtca cgactacgtc
  58381	ccgaccacac agtcctctca gccagcccga gaagcagggc cctcccctga cccccatctg
  58441	ggcctgggct tcagttttct cctccctgca atggggtgac tgtttgcctc caggagaggg
  58501	gagcatgtaa aggtggccac tctcttctgg cagacatgcc aggcctgggc cagcctccac
  58561	ccctttgctc ctgcagcccc tgctgacctg ctcctgtttg ccacaccggc ccctcctggg
  58621	ctgatcaggg cccccctcct gcaggaagcc ctctgggaca agcccagctt gctgtaactg
  58681	tggctttcca ctgtgacctg caacgtggga ggctgttact taaaactccc atgactggtg
  58741	gattgccggt ccccagaaca aggccacgca tccctggagg ccctcgagac catttaaggt
  58801	agttaaacat ttttacttta tgcattttca tgtgtatcag aaagaaaaaa aatgtatcat
  58861	cagttcatca aatccatgat ttcttgacca atattgctaa gatgaggctg aaataggcat
  58921	ttccattttt aaaaaactga atcactctga agaaacagat ggcaggcttc cctggtggtc
  58981	cggtggttaa cagtccatgc ttccagtgct gggggcatgg gttcgatccc tgaaaatttt
  59041	aaaaaggaag aaaaagatgg ctcccccgtc cctgggattc tccaggcaag aacactggag
  59101	tgggttgcca tttccttctc cagtgcatga aagggaaaag ggaaagtgaa gtcgctcagt
  59161	cgtgtgcgac tcttagcaac cccatggact gcagcctacc agactcctcc gtccatggga
  59221	ttttccaggc aagagtactg gagtggggtg ccattgcctt ctccaggcaa acggcctgct
  59281	actgctactg ctgctaaatc gcttcagtcg tgtccaactc tgtgcgaccc catagacggc
  59341	agcccaccag gctcccccgt ccctgggatt ctccaggcaa gaacactgga gtggggtgcc
  59401	attgccttca gcctgctgct gctgctgcta agtcgcttca gtcgtgtccg actctgtgtg
  59461	accgcataga cggcagccca ccaggctccc ccgtccctgg gattctccag gcaagaacac
  59521	tggagtgggt tgccatttcc ttctccaatg catgaaagtg aaaagttaaa gtgaaattgc
  59581	tcagtcgtgt ccgactctta gtgacccaat ggactgcagc ctaccagggt cctccatcca
  59641	tgggattttc caggcaagag tactggagtg gggtgccatt cggcctaggg agtgagaaat
  59701	cacggctgtc ttccctcttc tcgccctcta ggggtctctg tggagcctcc ctggagaggc
  59761	cgcggcggct ccggggactg gagggggagg gggggttgag tcagccggtg gccctcccct
  59821	cgctgcccgt ctcctccctt tttaggcaca agctgggcgc cctttttagg cgcagcctca
  59881	ccctgcgggc cactgcccgt gtttcggctc cccggagata aaacagattg cctgcacccc
  59941	gggtcatcac aaggattgta tgaccgtttc ccagtgtgct caccaccctc cctctgattc
  60001	tcagagacgc gccctcgcct caggaggctg ctcatcccag gccaaggggc ggcgtggggt
  60061	ccccagcgcc ccgcacagac actgccttct gaccacctcc tcccaacagc ttacctgcca
  60121	agaaggcctc ctgacccctc atcctgcccg gtggtttgga gaaagcctca tctggcccct
  60181	ccttctcggg gcctcagttt ccccctctgt gaactggcgg attctgccaa gctgacgtcc
  60241	tggccagccg cctccccgtg gccagtgtcc cccgggacac agctgaatgt ccctgctcgg
  60301	gatgcacctt cccaagttgg cctgtcagga ggcgggggcg agcagggaaa cccgactcct
  60361	ctcagacggc ccatcgcatt ggggacgctg aggcccggag cagcggcacc ctcctggcca
  60421	gggtcattct cccgccccgc cccgtccctc cgggcctccg agaccgcagc ccggcccgcc
  60481	ccgggaagga ccggatccgc gggccgggcc accccccttc cctggccgcg ggcgcggggc
  60541	gagtgcagaa caaaagcggg gggcggggcc ggggcggggg cggggcggag gatataaggg
  60601	gcggcggccg gcggcacccc agcaggccct gcacccccgg gggggatggc tcgggccgcc
  60661	ggcctccgcg gggcggcctc gcgcgccttt ttgtttttgg tgagggtgat gggggcggtc
  60721	gcggggtact attttttcat ttataattgg gtattagcta gcgagtggaa ccacaccctt
  60781	attccactat agccaatttt tgcgggggca tcttacatta cagactcgcc cgcctcttat
  60841	ttcggtacag catatcagat cgtctcttta ctcagacact agtgattatt gtctatagta
  60901	cacaaaaaga acggttgtgt cggcgtaatg gttgcatttt ccctcctcgt ttctcctgac
  60961	cacctcaatt acaccaacac tctactattt aaatcacgta ttgtacgcca ccctccgccc
  61021	gcgaactaaa agaatgtgca gatattctga agataaaatc gttcattgtt acgccccgcg
  61081	cgcttcgcgt atattactct tagaacttct tattcgcccg agcagttatt caccccccgc
  61141	aactagatgt cgccttaata tttgttctaa ccgttttgga ttctaacgat aggcgggaaa
  61201	ggtagacatt cgaccgctac gacaactaaa atcgacgagc acaggctatt tatatcgcga
  61261	ccacacgcgc gcggtataca naccgtaaaa ttatctaaca tcgagagtaa gggcacagag
  61321	cgaaatacaa gcggcgtggt gggaggtgtg tctgtagtga attcgcacct cgcgccgccg
  61381	cctctgtgcg tcgnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  61441	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnngatataa
  61501	tattaataaa cagcggatag atgtgtgtaa gggaggaggt gcataagaga ttaaagagag
  61561	gcgggcggag agaaatagag tagaggagga tgagagaaaa aagaaagcaa gcgtaggtac
  61621	aacggcgggt gggtagtatg ataaagtgag tgtatatatt tgagtaaagg aagggtagat
  61681	ggagtataaa gaagtaagga gaggagaggg cggcggagag agagagtgca aagaaaataa
  61741	gtgggcaaag gcggggtggg tgagaagcag tagaagagaa gatagagaag ggggaaaaag
  61801	aggaaaatga ggattagaac aagtaggaca ggatagatgt gaaaaatgag atcaggtcaa
  61861	ggtggagaaa aagtagaaac tggggcgtga ttgtaaaaaa gggaggccgc gatggggcag
  61921	caccataagc gaagagatga attaatgaaa gcaaggcagg gagaatcaaa tgagttgggt
  61981	ggaggaagga ggctgtgact tccttcgctg ccggaaagag aactagaata gcctcgggct
  62041	gtggggggag gtaaagataa agtgacttct gggccctggg ggaggcccag gagtttctac
  62101	cgagctgagc tgggtgcctc tcccaaatgc ccaaccccct gagagtcgac gggagagcac
  62161	agcctggcca aacctgggca gggcacacgt gtccttcacc ccacagtggt cacgagccca
  62221	gcgtggtccc tgcgtctggc gggaaacaca gaccctcaca ccccacacaa gggtccggcc
  62281	gctttcaaat aacagcagcc gtgccctctg ggccggtgac ccggacacag agagatgaag
  62341	tccgcatctc tcagagtgcg ctgtcctccg cccggtcagg cccgggtccc ctgcttctct
  62401	gaggtcacca ggagggattg catgtgggtc tcagggacac aggttcagtg atgtgacaga
  62461	gggtagtggg tcccagcagg gccggtcttt ggacccgttt ttctgaaaag ccagttggcg
  62521	acctggggtc acagcaaagc tgatcctgtt tggccaggag tctcccagtg acggcctccc
  62581	ccagaacatc gggcccagtg ggggctccag ggggtagact tgcctcccag ctcacgcccg
  62641	tgtcttgaca agtccatgat ttggtaaaat taatttgtgt tggatggagt tgatttagtg
  62701	gtgtgtgagt ttctgtggcg cagcaaagtc aatcagttac gcatacacat gtatccagct
  62761	cttcctacga ttctgttccc atataggtca ttatggggtg tcaggtagag cttcctgtgc
  62821	tacgcagtac ggccttattc agttcagctc agtcgtgtcc gactccttgt gaccccatgg
  62881	actgcagcac gccaggctcc cctgtccatc accaactcct ggagcttatt caaactcatg
  62941	tccatcgagc cggtgatgcc atccaaccat ctcatcctct gtcgttccct ctcctcctgc
  63001	cttcagtctt tcccagcacc ccctagagaa gggaatggca aaccacttcg gtattcttgc
  63061	cctgagaacc ccatgaacag tacggaaagt ccttattagt tttctatttt atatatagca
  63121	gtgcacacgt gtcagcccca atctcgcaat ttatcacccc cctccgccgc cgattggtag
  63181	tcatgtttgt tttctacatc tgcgactcta tttctgtttt gtaaacaagt tcatttacac
  63241	cactttttta gattctgcac atacgtggca agcccacagc aaacatgctc aatggtgaaa
  63301	gactgaaagc atttcctcta agatcaaaaa caagacgagg atgtccactc actccgtttt
  63361	tactcaacac agccctgaac gtcctagcca tggcaatcag agaagagaaa gaaattaagg
  63421	aatccaaatt ggaaaagaag aagtaaaact cactctttgc aaatgacatg acacttatac
  63481	ccagaaaatc ctagagatgc taccagataa ctattagagc tcatcagtga atttgttgca
  63541	ggatacaaaa ttaatacaca gaaatctcct gcattcctat agactgacaa caaaagatct
  63601	gagagagaaa ttaaggaaac catcccacgg catgaaaaag agtaaaatac ctaggaataa
  63661	agctacctaa agaggcaaaa gacctgtact cagaaaacta taaaatactg acaaaggaaa
  63721	tcagacgaca cagagagaga gagataccac gctcttggat gagaagaatc gatagtgtga
  63781	caatgactat actacccaga gaaacataca gattcagtac aacccctatc aaattcccaa
  63841	tggcattttt cacagaatca gaattagaac aaaaagtttt acaagtttca gggaaacaag
  63901	aaagatccta aagagccaga gcaatcttga gaaagaaaaa tggagctgga agagtcaggc
  63961	tccctgagtt ctgactgtgt atacaaagct ggcatgattt ttaacagcag gggtgtaaat
  64021	gaacttgttc acaaaacaga tggtggggtg ggcttccctg gtggctcagc tggtaaagaa
  64081	tcctcctgca acgcaggaga cctgggttcg atccctaggc tgggaagatc ccctggagaa
  64141	gggaaaggct acccactcca gtattctggc ctggaaaatt ccaaggacca tatagtccat
  64201	gggtttgcaa agagtcggac acgactgagc gacttccaat cctggaaacg tcccattgtg
  64261	gacggtgaac tggggttgtc caagctcagg gtaaccgttt gctgagtgac tgacactcct
  64321	tctcatgggt taaaatgtgg ggcccaaggc caggaccaga ccccgcagtc agccaggcag
  64381	accctgtgca gccccagcga gtgtgtggcc gccgtggagt tcctggcccc catgggcctc
  64441	gactggagcc cctggagtga gcccattccc tcccagcccg tgagaggctg ggtgcagccc
  64501	taaccatttc ccacccagtg acagatccgc ctgtgtggaa acctgctctt gtccccaggg
  64561	aacctggcag gactcaggga gaatgtctca gggcggccac agatcagggg ctgggggggc
  64621	agggctgggt ccagcagagg ccctgtgccc actccccgga aagagcagct gatggtcagc
  64681	atgacccacc agggcaccga cgcgtgcttg cacacaggcc gccccctcat ggtgacactc
  64741	ttttcctgtg gccacatctc gccccctcag gtccctcctg ctccccagct cctggcctgg
  64801	gaacctcttc cccgccccgg ggacgtcagg gctggtgtcc actgagcatc ccatgcccgg
  64861	gactgtgctg atcaccagca cctgcacccc ctctcgggtc tcaccaggat gggcaactcc
  64921	tgcccatcca gcacccagcc tcctgggtac acatcggggg aggagggaga agcctgggcc
  64981	agacccccag tgggctccct aaggaggaca gaaaggctgc cgtgggccag ccgagagcag
  65041	ctctctgaga gacgtgggac cccagaccac ctgtgagcca cccgcagtgt ctctgctcac
  65101	acgggccacc agcccagcac tagtgtggac gagggtgagt gggtgaggcc caggtgcacc
  65161	agggcaagtg ggtgaggccc gagtggacag ggtgagtggg tgaggcccag gtagaccagg
  65221	gcccatgtgg gtgaggcccg ggtggaccag agtgagcggg tgaggcccag gtggacaggg
  65281	cgagcgggtg aggcccaggt ggacagggcg agcgggtgag gcccgggtgg acagggcgag
  65341	cgggtgaggc ccgggtggac agggcgagcg ggtgaggccc gggtggacag ggcgagtggg
  65401	tgaggcccgg gtggaccagg gcgagtgggt gaggcccggg tggacagggc gagtgggtga
  65461	ggcccgggtg gaccagggcg agtgggtgag gcccaggtgg acagggtgag tgggtgaggc
  65521	ccaggtagac cagggcccag agcaaagccc cggctcagca gtgatttcct gagcgcccac
  65581	tgcttgcagg gacctcagcg atggtaaggc agccctgttg ggggctcccg actggggaca
  65641	gcatgcagag agcgagtggt cccctggaga aacagccagg gcatggccgg gcgccctgcc
  65701	aggctgcccc aggggccaca gctgagcccc gaggcggcca ggggccggga cagccctgat
  65761	tctgggttgg gggctggggg ccagagtgcc ctctgtgcag ctgggccggt gacagtggcg
  65821	cctcgctccc tgggggcccg ggagggacgg tcaggtggaa aatggacgtt tgcgggtctc
  65881	tggggttgac agttgtcgcc attggcactg ggctgttggg gcccagcagc ctcaggccag
  65941	cacccccggg gctccccacg ggccccgcac cctcacccca cgcagctggc ctggcgaaac
  66001	caagaggccc tgacgcccga aatagccagg aaaccccgac cgaccgccca gccctggcag
  66061	caggtgcctc cctctccccg gggtgggggg aggggttgct ccagttctgg aagcttccac
  66121	cagcccagct ggagaaaggc ccacatccca gcacccaggc cgcccaggcc cctgtgtcca
  66181	ggcctggccg cctgagacca cgtccgtcag aagcggcatc tcttatccca cgatcctgtg
  66241	tctgggatcc tggaggtcat ggcccctctc ggggccccag gagcccatct aagtgccagg
  66301	ctcagagctg aggctgccgc gggacacaga ggagctgggg ctggcctagg gcaccgcggt
  66361	cacacttccc ctgccgcccc tcacttggga ctctttgcgg ggagggactg agccaagtat
  66421	ggggatgggg agaaaaatgg ggaccctcac gatcactgcc ctgggagccc tggtgcgtct
  66481	ggagtaacaa tgcggtgact cgaagcacag ctgttcccca cgaggcctca cagggtcctt
  66541	ctccagggga cgggacctca gatggccagt cactcatcca ttccccacga ggcctcacag
  66601	ggtccttctc caggggacgg gacctcagat ggccagtcac tcatccattc cccatgaggt
  66661	ctcacagggt ccttctccag gggacgggac ctcagatggc cagtcactca tccattcccc
  66721	acgaggcctc acagggtcct tctccagggg acgggacccc agatgggcca gtcactcatc
  66781	catccgtctg tgcacccatc cgtccaacca tcacccttcc ctccatccat ctgaaagctt
  66841	ccctgaggcc tccccgggga cccagcctgc atgcggccct cagctgctca tcccaggcca
  66901	gtcaggcccg gcacagtcaa ggccaaagtc agacctggaa ggtgcctgct tcaccacggg
  66961	aggagggggg ctgtggacac agggcgcccc atgccctgcc cagcctgccc cccgtgctcg
  67021	gccgagatgc tgagggcaac gggggggcag gaggtgggac agacaggcca gcgtgggggg
  67081	ccagctgccg cctggctgcg ggtgagcaga ctgcccccct caccccaggt acaggtctcc
  67141	ctgatgtccc ctgccctccc tgcctccctg tccggctcca atcagagagg tcccggcatt
  67201	ccagggctcc gtggtcctca tgggaataaa aggtggggaa caagtacccg gcacgctctc
  67261	ctgagcccac ccccaaacac acacaaaaaa atccctccac cggtgggact tcaccagctc
  67321	gttctcaggg gagctgccag ggggtccccc agccccagga agccaggggc caggcctgca
  67381	agtccacagc cataacacca tgtcagctga cacagagaga cagtgtctgg tggacaggtg
  67441	cccccacctg cgagcctgga gagtgtggcc ctcgcctgcc ccagccgcgg tcagtcggct
  67501	cagcaaccgc tgtccactcc cagcgccctg gcctcccctg tgggcccagg tcaagtcctg
  67561	ggggtgaagc taagtcaggg agcctcatcc atgcccagcc cggagcccac agcgccatca
  67621	agaaatgctt cttccctcca tcaggaaaca ttagtgggaa agacaagagc tggggggttc
  67681	tggggtcctg ggggatcaga tgaaggggtc tgggagcagc agcagcctca ggcaccccaa
  67741	aacaaggccc aggagctgga ctcccagggc tgaggggcag agggaaggaa ggcctcctgg
  67801	ggggttggca tgagcaaagg cacccaggtg ggggctgagc acccctcggc tggcacacac
  67861	aggcccccac tgcagtacct tccccctcgg agaccctggg ctcccgtctc ccgcctggcc
  67921	tgccatcctg ctcaccaccc agaaatccct gagtgcggtg ccatgtgact gggccctgcc
  67981	ctggggagga aggagattca gacagacagg atgccagggc agagaggggc gagcagagga
  68041	tgctgggagg gggcccgggg aggcctgggg ggcagggggg caggagttct ccagggtgga
  68101	cggcgctgtg ctatgctcgg tgagcacaga ggccccgggt gtcccaggcc tgggaaccca
  68161	gcagaggggc agggacgggg ctcaaaggac ccaaaggccg agccctgacc agacctgtgg
  68221	gtccagaagg cagctgcgcc ctgaggccac tgagtggccc cgtgtcccga accaccgctg
  68281	aaacatggga cacacgttcc caggcggagc cactcctgcc ttccgggagg ctcccagcgg
  68341	gctcatcgct ccatcccaca gggagggaaa ccgaggccca gatgacgaac atcccggcga
  68401	gcaggtcaaa gccagcccct ggggtcccct ctcccggcct ggggcctccc ctctgcaggg
  68461	tgggaaaccg aggccacaca ggggctccat ggggctgccc tctgccaggc cctggacacc
  68521	ccgcgggtga cccccgcctc tatcatccca gccctgccag gccctggaca ccccgtggat
  68581	gacccccgcc tctatcatcc cagccctggg ggacagatgg gaggcccaag cgtggacccc
  68641	ctggccaccc cctaccccac agccgggagg agccgggagc tggtggccaa gggcctagag
  68701	gagccagann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  68761	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnca atatagaggg
  68821	ggtgggataa agggtaatat gatgtttagg tagttagagt taaattagaa gggtttggat
  68881	aaagattaat aaaattacaa gcgtacatat cgtgtgagtg tgggtgataa tatttgtgta
  68941	tgtggggaat agaagtgagt gtgagtagta ttcaagatgt aagtgtgcga atacaggtct
  69001	gagcgatttg aatggaagtg aaaaaaagcg tgtgtgtgga ggaggcggga gaggaagata
  69061	gtgtggggga agaaaagaag gctagtgggt aaagaaatat cagtaggcgg ttgacgaaag
  69121	aagaactagg aagaattaat ataaaaataa agggaggatt aaaaaataaa gagggaggag
  69181	gtaacggaaa tagttagtta agaaaagaat ggagagtgga ggtaagataa ataagggagt
  69241	aatgggagtg aggaggaata aataaaaaaa tggtgaggga aaatagagta gaatgagaac
  69301	aagaatgaaa aagggagtga agggggtgaa aaaaagtgaa gttgaaaaaa gaggaaaaaa
  69361	aaggagaaga taaaaaaata aaataaaaaa aggaaaaaaa agaaaaaaag aaagaagggt
  69421	taaaggacga aaagaaggga agagaaaaaa aatagtttaa gtgggggagg gtaaaaaaga
  69481	attaataaag taaatatggt tgtggtcgaa aaaaaaaaaa aaattgttgt gttgatgaga
  69541	agaaaagaaa aaagaagaaa gggaaaagca aaaagaaagg agagaaaaag acaaccccac
  69601	cgcccgggcg catggagggt gaggatggcg cacgcccgcg gatggcacag catcacagca
  69661	atcctaaaac gttttcagac cggtgcatct tcaccgcgcg cgcgccccgc ccggccctcc
  69721	tcccgccctg accgcggacc cccacccgca ccggggagcc tacccccacc ccggggacgc
  69781	tccgccacgc taaggtcagg actgccgtga agacgcgccg gggtgaaaac gttttatctt
  69841	catgacataa gcgagtggtt ttgaaacagg tttacaaacc ctcgtgaaga cgcaccctta
  69901	gcgttaggtt ttgttttttt accatgtgac gatgcaacta ttttcttcct ctcttccaca
  69961	gtggctagtc gcctccagag cgaggggtat ctcttgtaca gagaccctcg gaacatccgg
  70021	aggtagtttc ccacctaggg gtaaagcgag aaggctcatt acgagggccg gggctcctcg
  70081	gggaagggca gggccctggc gcagaggctc tgccacctca gtgacacgca gaccacgcgc
  70141	ggcctgcagg cgccgggctc tgaaagcagg caaagcccga tctgctgaca tcaggggttc
  70201	cgcagcagcg aaggtctggc ccgcacctgg cccactggca gggggtaagc tctgcctccc
  70261	gacgacagca ccaagttcag gaagggccac gcagacactg gtgagacacg gcccccccgg
  70321	agctgcccga gaagctctga ctttgcacta aagatctctg gcgcggtcca aaaatgtaag
  70381	gcctctcttc cttttatctt aagactttga tatttttacg atgtaataaa taccaagaag
  70441	ggcttttaat ttcagacaga tgtaggataa tttcccccgt agcccttgct gctttgttta
  70501	gtaacgaaac tcaaaccaga aataccaaag gaattttcca aagagtttca aaagcgctta
  70561	tcagcaatca ctagactgct gcatacatca tcactgcccc aaacaatagc ctgcctgtgc
  70621	cagttactca aagtactact tacttgacga aaacaaatct agtcctaacg tttttacaaa
  70681	gaaactccac tcttccgcca acttttcaga aacaaccact cgatcacgtg gcaggggacc
  70741	gtggctggac tgggtgctgg ctccttctgt gaccaggcaa cactgccccc ttctcggcct
  70801	ccctacgcct cttgacaaat gttcatcagc tgtaaagttc accccacgag ggacccactt
  70861	ctgctatttc ccacgtacct accccattat aggagttttc tttgtgacag tttctgcatt
  70921	tttcatggat ttagaggttt acataatcag ggctgctgaa cagcatgaga gacgtggcca
  70981	caaggtccct cctgcacctt gccgcagggg cagggcgagt tatctggctt gagcgtggtt
  71041	accatcaggg ggtaaacaca gtttccagga cgtttttgac aagacactga cccggatgcc
  71101	cccactacca ccgtgcaggt cctgcaggcc tcccagcctc ccaggccctt cccgaggtcc
  71161	cttcggaact taggggactc ggtctgcccc cctgggtttt ccctgcacca gcttttgccc
  71221	cctctggacc caggtttccc aaatggaaaa cgaaggtgtg ggtatggaag ctccctgggc
  71281	tcctctcagc tgtgcctctg catggtgatg acggctgccc atcggggggg gcaggactgg
  71341	ggcagctgcg gacaccctcc caaggctgct acccccgagt ggtgtggggc gctgtgggca
  71401	cgctctgctc agcgcacctc ctggaaacca gcgcctgccg tctgcccggg gcaaccggcc
  71461	cgggagccaa gcaccactgc cgtcagagga gctgctggct gtgagtggac gccagtctag
  71521	ctctgaaccc tgcccaggcc tcctgaggtc tgaacattgt aaaatcaggc cccggacggc
  71581	aactgcctct ccctcctgcc gtctggtctc cataaactgc atctcaggac aaatcttctc
  71641	actcaccagg gctgaaacag aagactgcag ctatctttct caaatctaag gtgtgctaca
  71701	gggcaagtcg cagaaactgt ctggcctaag catctcatca gatgcctgag acaagagctg
  71761	tggacgccaa gctggagcca gagctcctcg cgttctgccc acctggcacc gcgttccacc
  71821	cagtaaacgc aggcttgatt ttcaaaagta ccaccgactc agagccaatg ctaaaccgac
  71881	cacttttcct gcccattaga ttgggtgaag gtttctttaa tcaatctgcc agtcaccaca
  71941	tgccgcctct gtgcccacag gctggcgaag acctttctga gctacggcat gtggcaggca
  72001	gcggcacctc tcttcagtac ggccagctgt caaggggagc gtttctgtga tgatgtgaaa
  72061	atacattgca tccggccccg tgtttcatga acacgggtga ggaaaggaaa cacacaaagt
  72121	tctgatgcga ctgacagcac gggtctcata actcaataca agtcagacaa accacaggga
  72181	gtcacaggga atcccaatag cctcatctag tgtgaccatc atgaggctta atttattcag
  72241	tgtattcaat cataaagagg gggaaaaatt gtaaaaaaaa aaaaaaagaa agagtgaaat
  72301	gtgtaatact gaaaactgtt gctaggagaa gcaagcattg gcgtttgtaa ctgctttgac
  72361	tccccaagac ccacactcgc ctcgctacaa aagggaggca ctgctgctca gtacttgcac
  72421	acccgaactg cggatttgta atttaaaaat gtgtgtgtgg acacagcaca agccagagac
  72481	tgccaaaggt tgagggacac tggaagaact taatatactt ggtgcatgct gccagtgaca
  72541	gtcagtcacc agctgattca atagagtgcc gaaaggtcac cttttaggta aggatgaagg
  72601	ggttctgggc tcgtttactt gcactaactc agagttagtc cgagatatcc gaagtgccag
  72661	gtgcctccca tttgctgatg gatctagctc agggacggct gggccctagc catccaaaaa
  72721	tcaagcattg ttctcccaac ctgtcttctc gctgataatg gaaggtcaga acgcccaccc
  72781	gcccacctca aagtcaaaga acaccaagcg ggtgagtccc cactaagctc ggtgtttcca
  72841	atcagcggtt tcaggattcc agctggggca atgagggagg gagcgtgcga gggatccaac
  72901	acctcgcccc gtgcgcagca agggataacc caacaccccg tttctgtacg tccggctgga
  72961	gttgtggaac tcagcgcgga cccggggcca ccgcgacccc cgggaccctg gccgcgcggc
  73021	gcatccccgc tgccgggaca cgggtaagcg tccccaaact gccggacgcg gggcggggcc
  73081	ttctccgcca cgccccgata ggccacgccc aaggacaagg atggtcgtgc ccagacggcc
  73141	ggggcgggnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  73201	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnncg gagggggggg
  73261	ggcggggcgg gggctgccgc cgcgcgtata ggacggtggt cgcccggcct ggggtccggc
  73321	cgggaatgac cccgcctctc cccgcatccc gcagccgccc cgccgcgccc tctgccgcgc
  73381	acccgcctgc gcacccgccg ccctcggccg cggccccggc ccccgccccg tcgggccagc
  73441	ccggcctgat ggcgcagatg gcgaccaccg ccgccggagt ggccgtgggc tcggctgtgg
  73501	gccacgtcgt gggcagcgct ctgaccggag ccttcagtgg ggggagctca gagcccgccc
  73561	agcctgcggc ccagcaggtg agcaagggct caggggaaac tgaggcccga cacagagccg
  73621	cagcaagaag gatcctactg gtcactcggc tgttggcctg gggtcatcac aggcgggctc
  73681	tcccaaccca tcccctgagg ccaaggtccc tagaaccccg tgggcagaca ccaaccagcc
  73741	ctttaaatat ggggaaacca aggtgcttag gggtcagaga tagccctagg tcgcccaacc
  73801	ctagtagaag ggagggctgt tggagttcct gagtgcccgc tctcccaccc cccgggaggc
  73861	cccttcctga gcccaagggt gactggtagt cagtgacttt gggcctgccg acctgtaccc
  73921	cactgggcac cccaccagtc ctgagccaca tttgggctta gtgacggggt cagggatcat
  73981	gaggatcaat gtggctgagc caggaaggtg ttagaacctg tcggcctgga gttcatacca
  74041	gcactgccct gggcttttct agacccatgt cccgcctcct gccccacctg cccctgttcc
  74101	cgcaccccac cagcagcggc aggggcttcg agagggctgt gggctcaccc tatttcaggg
  74161	atggagccgc taagacctgg ggcacactgc ccgctaggga cccctgaggc accagggccg
  74221	ggggctctgc ggaggggcag ccgccacccc cagctttgga gtcctctccc gggtgcccag
  74281	cccgagctga tccggctgcc tcccacgctg tgccccaggg cccggagcgc gccgccccgc
  74341	agcccctgca gatggggccc tgtgcctatg agatcaggca gttcctggac tgctccacca
  74401	cccagagcga cctgaccctg tgtgagggct tcagcgaggc cctgaagcag tgcaagtaca
  74461	accacggtga gcggctgctg cccgactggc gccagggtgg gaagggcggt ccacggctcc
  74521	cactccttcg gggtgctccc gctattccca ggtgctcctg cacttcccat gtgctcccga
  74581	ttctccctgg tgctccctct cctcctggct gctcctttgc ctcccaggtg ctcccacttc
  74641	tccctggtgc tcctgctcct cccggcggct cctgtacctt cggcctgacc tcctccctct
  74701	acaggtctga gctccctgcc ctaagagacc agagcagatt gggtggccag ccctgcaccc
  74761	acctgcaccc ccctcccacc gacagccgga ccatgacgtc agattgtacc caccgagctg
  74821	ggacccagag tgaggagggg gtccctcacc ccacagatga cctgagatga aaacgtgcaa
  74881	ttaaaagcct ttattttagc cgaacctgct gtgtctcctc ttgttggact gtctgcgggg
  74941	ggcggggggg agggagatgg aagtcccact gcggggtggg gtgccacccc ttcagctgct
  75001	gccccctgtg gggagggtga ccttgtcatc ctgcgtaatc cgacgggcag cgcagaccgg
  75061	atggtgaggc actaactgct gacctcaagc ctcaagggcg tccgactccg gccagctgga
  75121	gaccctggag gagcgtgccg cctccttctc gtctctgggg gcccctcggt ggcctcacgc
  75181	tctgtcggtc accttgcccc tcttgctgat gcaatttccc cgtaattgca gattcagcag
  75241	gaggaatgct tcgggccttt gcacctgacc gcatgagcag aggtcacggc cagccccctt
  75301	ggatctcagt ccagctcggc cgcttggccg tgacgttcca ggtcacaggg cctgccggca
  75361	cagaggagca ggcccttcag tgccgtcgag cactcggagc tgctgcctcc gctgagttca
  75421	ctcagtgtct acgcacagag cgcccactgt gtaccaggcc ctattccacg ttccccagtc
  75481	accgagcccc cagggctggt ggggacctgc cctcgggtac actgtgtccc gtcacgtggc
  75541	tttacgtgtg tctctgaggg aggctggcat tgcggtccac ctctcagcac aaacatctgt
  75601	cccctgggaa gggggtccca tttctgggtg cgagcagccc cctggggtcc gtgtctcctc
  75661	cttacctggc tcaaggcccc ggctcctggg tcctggacag cagggagccc acccctcggg
  75721	gctgtggagg gggaccttgc ttctggaggc cacgccgagg gcccaggcgc cgcctccggc
  75781	cgtcgccctg agggagcagg cccgacgcca gcgcggctcc tctgtgaggc ccgggaaacc
  75841	ctgcctgagg gtgcgggtgg gcaggtgccc ctgcccccag gctctcctgt gtgagtgaca
  75901	ctcaccagcc agctctggat gccacccatc cgggttctcc aggaggcact catagcgggt
  75961	ggggtcccct ccctcccccc tctgtggagg gagggagtct gatcactggg aggctggtgg
  76021	tccgtacccg cccccccgac tctggacgtg tttactaccc ccgcctgggc tcaggacagg
  76081	gcattggatg ggaaggacag ggctgggtcc tggccaggct gggggctctg cagggcatgg
  76141	gtgcccctgt ctcttcttat attccaacgt cactgcaggg gggcgcaaat cttggacccc
  76201	acttactgat gatctgcatc aggacatagg tcccccctcc tgcagcgggg ggctggccac
  76261	ggagggcgct ggggaaggcc cctcctccag cccctcggcg aggctcacca ggtgcccatc
  76321	ctcagccagc agggcgacgc tcgctgggag ggcggagagg gaggcagggc agggctggta
  76381	cgacccccgc tggggcgggg gggccctcag ccggtcctcc agcacccttg ctgccccccc
  76441	tcaccgtcag ggggcacctg gccgctctgc ctcaggtggg cggtgagggt cccaaggcca
  76501	caccaggtgt tcaccagctc ccagcagctg gctgtgggag aggggcagag gtgggcgcat
  76561	ggcacccgcc ttccccccag accaggatgc tctgccttcc tcccgcccat ctccccagac
  76621	atctgaagga ctcttgcctc caccatgcag ccccgcctcc accagaagct caggttcccc
  76681	gccccccctc cccgaagctg caggacccct gaccagcgaa gagatgggac agttggaaca
  76741	cacgctcccc cagcagcggc acagcagctg tgtggcccag aagagcccgc ctgtttccct
  76801	caagcaactc cccatggatg tcatcccatg gacaccccct tccccacacc gcctcctcgt
  76861	tctccccctc caaggcagag ggaacgcacc cccacctgtc tgctaggaca ggggacccca
  76921	cttacctccg aacatcacct tgataaacat ggccgtggtg gggacagatc cctccgaccc
  76981	ccaacttccg acctggggaa ggagctgggg tggagctcga ctgcagggtg gggccctgtg
  77041	ggaggtgtac gggtggagag ggtgatgggt gggtgggctc aagcggagct ccttgctcag
  77101	tccaggcggt ccctgcagct agtccaggat cctcagcctt ctccccctca ctggatcagg
  77161	gaagactgag gttccctccc ctgccccccc acccagcttc caagctggtc tctgtggcag
  77221	tgggagctgc caagaggtct gagcggccag tatccgggta acggggtttg tggagggtcc
  77281	gggcattccc ggtgcagggc tctagtgggg gctggagcct cgggcccaga gctgtccaga
  77341	gaccagtgcc ctcccaccgc cgccgcccgc aaggagagac agagctccca ggcggggagt
  77401	cggaggttcc tggaggggga gcatcctcaa ctctgcaggc ccccttccca ggcgcactcc
  77461	cggcctcccc gtcttctgtc ccctgctctt gttgaagtat gattggcata cagttcacag
  77521	ccactcttcg gagtgttctc cacactaagg atacagaaca tgtccctcgt ccccccaaac
  77581	tcccagccag gctgtcacga agagggaggc ggccgacggg gcagggcctt gcactcctgc
  77641	gtgtggggtc cacaggggtc gtccccgtgt cggtggcccc ttcctctcac gccaggaggg
  77701	tccccttgcc tggaggtgcc gtggatccgc tcgctgcctg ctctttgggt tgtttcccgc
  77761	atggggtgat gatgaagagg ccagtacaga cactcgccag caggtctctg ggtgaacagg
  77821	catttatttc tctttcctga gggcagatcc tgggagtggg gtgccggacc gtccggggag
  77881	agtatgcttc tgtttctaag aagctgccgt gttctccagt gtgctgcacc atgtcacggc
  77941	ccctctgtgc gtctggactc aggagacctc cttctcagcg gccctccccc ccaggtggtc
  78001	aggccatctg tgcccttctg ggggcagagc tcagcgccgg aggcgggagg aggcccagat
  78061	cccagcgcag cccaccagcg ttgctctgct tccctcggca ttcatagctg gagaaagggc
  78121	aaggagcacc ggctgaagcc ccacctggag gacgcacttc gatggcagca ggtgctcaga
  78181	ggtggccccg ggcagcattc cccagacgca caggccagtg ctttcttccc aggacaccac
  78241	tgtgtctggg gacccgagtc ctgcagcacg gtcgggagcg gctgtgccca gattccggcc
  78301	tgcacccttg gctccagcca ccacccctgt ttgtcaaggg gtttttgtct ttcgagccgc
  78361	cgaggaggga gtcttttgtc tgcagtgtca cagaagtgcc ataaagaggg gcccacagtg
  78421	ggagctttat aacattggtg cggagggctg taacaggtca gggaggcact tgagggagcc
  78481	ttctagggcg atggagatgt tctaaaattt ggtctgggta caggctacag agatgtgtgg
  78541	gtgtgtgtgt gtgtgtgtgt aaaaccctcg agccacacgt gtgaggtctg tgcatgtgac
  78601	cgtacacagg agacctcggt ggaaagcagc cacctgctct gactgcacct gtggatttcc
  78661	agctcctgcc ctcaggcggc cctgcggggc ccactggctg acggggagac ggcaccgccc
  78721	tcccccgctg tcagggtggg ggggctgacg atttgcatgt cgtgtcaggg tccagcggcc
  78781	tcccttgcgt ggaggtcccg aagcacctgg agcgccgccc gcagaacagc ggactcctgc
  78841	ctgcctccct gcctctggcc atggcctgcc cgcctctggc cctctttctg ctcggggccc
  78901	tcctggcagg tgagccctcc caaggcctgg ctcacctagg ggtgtgtaag acagcacggg
  78961	gctctagaag taaatcgcgg ggaagtaaat cgtagtgggc aggggggatg gtttccgaag
  79021	gggccctgag ggggacagga gacctggcct cagtttcccc actggtgagt gaccagatag
  79081	ccagggtacc tttggactct gactctgggg ggctctcaga gactggtctc ctactcagtt
  79141	tttcagaggg gaagctggtg tggccttgtc actgccctgc agggcctcag ggacaagcta
  79201	tccctgagga ggtctccagc agtcagtggc cggaggctga gccgatggat atagtaacag
  79261	cccaggcggc ctcttggggg tggtcagcct gtagccaggt tttggacgag ccgaagtgac
  79321	ctaagtgatg ggggtctgca gagcaaggga tgagggtggg cagcaggagg acccagagcc
  79381	caccagccca ccctctgaat tctggaccct tagctgcatg tggctccttg ggaagacggg
  79441	gcttaagggt tgcccgctct gtggcccaca cagtgctgat tccacagcac tggctgtgag
  79501	cttttgggag cagattctcc cggggagtct gacccaggct ttgtggggca ggggctggag
  79561	ggaaggggcc caggccagac ctgagtgtgt gtctctcagc ctcccagcca gccctgacca
  79621	agccagaagc actgctggtc ttcccaggac aagtggccca actgtcctgc acgatcagcc
  79681	cccattacgc catcgtcggg gacctcggcg tgtcctggta tcagcagcga gcaggcagcg
  79741	ccccccgcct gctcctctac taccgctcag aggagcacca acaccgggcc cccggcattc
  79801	cggaccgctt ctctgcagct gcggatgcag cccacaacac ctgcatcctg accatcagcc
  79861	ccgtgcagcc cgaagatgac gccgattatt actgctttgt gggtgactta ttctaggggt
  79921	gtgggatgag tgtcttccgt ctgcctgcca cttctactcc tgaccttggg accctctctc
  79981	tgagcctcag ttttcctcct ctgtgaaatg ggttaataac actcaccatg tcaacaataa
  80041	ctgctctgag ggttatgaga tccctgtggc tcggggtgtg ggggtaggga tggtcctggg
  80101	gattactgca gaagaggaag cacctgagac ccttggcgtg gggcccagcc tccccaccag
  80161	cccccagggg cccagactgg tggctcttgc cttcctgtga cgggaggagc tggagtgaga
  80221	gaaaaaggaa ccagcctttg ctggtcccgg ctctgcatgg ctggttgggt tccaacactc
  80281	aacgagggga ctggaccggg tcttcgggag cccctgccta ctcctgggtg gggcaagggg
  80341	gcaggtgtga gtgtgtgtgt ggggtgcaga cactcagagg cacctgaagg caggtgggca
  80401	gagggcaggg gaggcatggg cagcagccct cctggggtag agaggcaggc ttgccaccag
  80461	aagcagaact tagccctggg aggggggtgg gggggttgaa gaacacagct ctcttctctc
  80521	ccggttcctc taagaggcgc cacatgaaca gggggactac ccatcagatg nnnnnnnnnn
  80581	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  80641	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn agagggtggg tgggtggaat ttaatatagt
  80701	ggtgcgcgtg gagcgtgggc ggcgcattta aggcggtcat ctaaaatagt ggataggggg
  80761	tggtgtgaca ataacgggtg gtggatgtgg tttacggggg gtgcaatagt tctgagtttg
  80821	ttagtgtctt cttgatgggg ttgcggcgtg tggacctacg ccttgagtat gtgggggggg
  80881	aaaagcagtg agggtagtag ggatgggaaa tattggtgga ggttctttgt tggtgtattt
  80941	tttggtatta tgttgggtgg tggagtggtg ggttgggtgt aatttcgctt gcgttatgtg
  81001	ttttttttct ttttcgtgtc gtgggttggg ttggttggtg ctttgtggtg gtggtgggtt
  81061	gtggtataaa aaaaaatgtg tggttgtgct cagcttagcc ctataacggt cggctttgtt
  81121	tcttgtttgt tctgtgggcg tgagcggatg gctcgggcct ccgtgctccg cggcgcggcc
  81181	tcgcgcgccc tcctgctccc gctgctgctg ctgctgctgc tcccgccgcc gccgctgctg
  81241	ctggcccggg ccccgcggcc gccggtgagt gcccgccgtc ctccagcccc cccgccccgc
  81301	cccgccctcc acgccgaggg gcgccggctc gcagagctgg atccaagggg gtgcccggga
  81361	gtggcccggc gcggcccgtt accccgaaac gctgtctggg tgccccgggg gtgtggtgga
  81421	tagtgagctt cccgtccctg gaagtatgca agtgaagccg gcgccgggat cgctcgggct
  81481	ggctggtgag cgggcgggac tcggtcgggc gctagacgca cgccgccagc cccccagctc
  81541	ccagacctgc ccactccgcg cccgcccggc cgcgatcccg ggtgtgtgtg tgtgttgcag
  81601	gggagggaca gcgggagtgg ctacagggct cccgactcac cgcagggaca aagacccgcg
  81661	ggtccccagc tggcgtcagc cgccaggtgt gtggcctcgg tgagcacacc tccaggcggg
  81721	agggttgagg gaagcgctgt ggggagggca tgcggggtct gagcctggaa gagacggatg
  81781	ctaccgcctg ggacctgtga gtggcgggat tgggaggcta tggaatcagg aggcagccta
  81841	agcgtgagag ctccggtgtg gcctggcggg ggtggtaggg gggggacgcc cctgtgtgtg
  81901	ccagcctgcg tgtgccctaa aggctgcgcc ctcccccact gctggggctt cgggggacca
  81961	gtcacagcct aggctactgc aggcgcacag ctccccggga gcccggccca cgcgggtgtg
  82021	ccgctgagcc tccagcctgt cggggcaggg gtggggggca gggatggggt cgttagcggg
  82081	gttgggggca gacgcccagg cagactctct gggcacagct ccggtgacaa gggaggtctg
  82141	gcaagcctgg gccccttctg tccagccacg ccagctctgc cctggccagt cttgccccct
  82201	ggcagtgctg gggatggaag ggggagcggg tacctcagtc tgggggccct gcctcctccc
  82261	cagccccgcc cggcccccta ggcctagggg cagagtctag gggtcaccct ggggagctgc
  82321	tgaatccgcg ggtttaggaa ccggagggac ctgggctttt gaaccacgtg gccctaggtg
  82381	agccctccgg cgcctcggta gccctcaccc ccagccttgt ccaggtgggc gggtgggagg
  82441	cgacagtgcc cactgctggg ctgaacagcg tctgcaggga ggccaggaga gctgggcaca
  82501	cggacacgtt ccatcacctg gagctgccac tgtgccactt gtgcggggtc aggcggggtc
  82561	tgagccgggc tgtcatctgt cacgccacag atatgcaggg ggcactcggg gtcgcctcgg
  82621	acatgcttat ccctggacgg ctgttggcag ggccgggaag gctctgtaaa tatttatcca
  82681	tcccagctca cagctttcag ggttgatgaa agccccgccg cccgcccact gtgggggacc
  82741	ccgccttccc ttctggagcc agcggggtga gggggtgggg gagatggacc tgcctgccca
  82801	ggagcaggcg gtgtgactct ggcaggtcac ttgacctctc tgagcctcag ggagggcccg
  82861	ggatggtgtg cggatgctct ctgccttcct cccagcctga ccagtgtcct cccctcgggg
  82921	tcgcctcctg cccaccgcag agggggtggc tatggggacc tgggccgatg gcaggcaggc
  82981	cggagagggc atgcccggct cagccgtgcc cagcacttcc cagtccaggg gcccccgcca
  83041	ctcccagccg ctggctgcct cccattttcc cgattgcagg ttggccccga ggctgaccgg
  83101	agcctctggc tcagctggga gactgaattc cccaagcaat tcctcaagga tgtgtgaggc
  83161	tgtggtgtgg tgcctatccg ggagaggtgg ggtgagcgga ctgggcacct ccgcccaggg
  83221	caggcccagg gagacgctgg ctgacgagca ggcaggcctg caaggaggac gagcagccat
  83281	ctcaggaatg tgggttttgg agacaagcca cagctggggg ggtggggggg ccatgggtgg
  83341	ggaggcctga tccccaggtc taggtccagc tctgggctcc ctcgccgtgt gaccctgggc
  83401	caagacctgg acctctctgg gccccgtctc ttcccctggg aggtggggcg atgcctgctc
  83461	cccaatcccc cagggctgtg gatgaggcag acgaggtgtg tgctcatccc cacctcactg
  83521	ccttccagca gccccgggcg gggggggtgg tggggactgg cgcacccagg tgaggatcag
  83581	gccttggagc tagggagggc cccccagccc caggccagaa aggacacggg gagacagaat
  83641	gcaggagggc ggcagagcag gggccagcgg tggggaaact gaggccaaga gcctgtggac
  83701	gatgtgctcc aggaaaggac ctcgctgcct ggggcctgga tcctagagcc tccaggagcg
  83761	gtgaccatga cgtgggcagg gaaccggagg ccccggcttg caggtggacc cggcgcgagt
  83821	cactcttcct ctctggccct gagagcttcc ttccagctgc cgctcctgtg ttctaatgtc
  83881	aagtctggag gcctgggggg caggtggggg ctgactgcca ggtgggggag ggcaggaatt
  83941	tggcagagca gcgtcccaga gtgggagaag ccagcccatg gaggggactc tctccatgcc
  84001	tgctgcccca aagggcgtta tagagagagg tcggttaccc cttcgccatg gccccgttcc
  84061	cattgaacag atgggaaagt ggaggctgag agaaggctgt gacttgccca gggtctccgt
  84121	ggcatggaac tgggcctgct gagtctcagg ccggggatct cgctgctgca ctgagcacgc
  84181	caggatgcag gggtctgggc ctggacctag cgcctcgtgg gggcaagaga ggaaggcacg
  84241	ctgggcctgc ctgtcaccct ccaccccacc gtggcttgtt gctcaggcct tcctgggggc
  84301	agaggagagg ggagatttca ctcgctggca ggctaggccc tgggctctct ggggctccgg
  84361	gggaacaatg cagccctggt ctttctgagg agggtccttg gacctccacc agggttgagg
  84421	aaaggatttc tgttcctcct ggaggtcacg gagccgacat ggggaggagc aggggcaggc
  84481	ccggggccca catcctcagt gtgagacctg gacgtgtgtc ctcccacctg acgctggggg
  84541	tggggggtgg gggccggggg ggatccagtg aaccctgccc ccaaattgtc tggaagacag
  84601	cgggtacttg gtcatttccc cttcctcctc ttcgtttgcc ctggtgggga cagtccctcc
  84661	cctggggaag ggggacccca gcctgaagaa cagagcagag ctggggtcag gggtgtgctg
  84721	ggagcgcaga gagcctcctg ctctgcctgc tggtcattcc tggtggctct ggagtcggca
  84781	gctggtgggg agcggctggg gtgctcgtct gagctctggg gtgcccaggg cctgggagag
  84841	ttgccagagg ctgaggccga gggtggggcc ctggcggccc ggctcctgcc ccaaatatgg
  84901	ctcgggaagg ccacagcggc actgagcaga caggccgggc cagacgggcg ctgaggctcc
  84961	cggcctctcc cccagctccg ctgtgaccct cacctgcggc ccggggtgcc agggcccccg
  85021	cttggttctg ccgtgtcttt gcaggctgat cccacgggct ctccctgcct ctctgagctt
  85081	ccgccttttc caggcagggg aaccgcgacc tccaggctgg gacgcgggga gggtgtatgc
  85141	gccaggtcag aatcacccct ccaccgggag agcgtggtcc aggggccctg gcagggtggg
  85201	gaccgagcat ctgggaactg ccagccaccc ccacccatgc agaggggaca tacagaccac
  85261	acggaggctg tgcctccgct gcagcaactg gagaacaccc agccgcggcc aaacataaat
  85321	aactaaataa taaaagtttt aaagatcgtt acttaaaaaa acaagtgtgc cccagtgatc
  85381	ggaccccagt tcccggtgcc ctgagtggtg ccggccctgt gctgagcatg gcctggttgg
  85441	ttcaccccca gatccacact aaagggtggg atcaccccta ctagtcaggt gagcagatgc
  85501	agggggggag ggcggcagcc cctccatgct ggtgggtggc cgtggtgggt gtcctgggca
  85561	ggagccagct cacggagctg gagaggacag acctgggggg ttgggggcgc ccaggaagaa
  85621	acgcaggggg agaggtgtct gccgggggtg ggggtccctt cgaggctgtg cgtgaagagg
  85681	gcaggcgggc ctgcagcccc acctacccgt ccccggccca aacggcggga gtaagtgacc
  85741	ctgggcacct ggggccctcc aggagggggc gggaggcctt gggatcagca tctggacgcc
  85801	agtcagcccg cgccagagcg ccatgctccc cgacggcctc cgctggagtg aggctgcgct
  85861	gacacccaca ccgctgaccc gggcctctct cccgctcagg atgccccccg ccgccacccc
  85921	gtgagcagag ggccacagcc ctggcccgac gcccctcccg acagtgacgc ccccgccctg
  85981	gccacccagg aggccctccc gcttgctggc cgccccagac ctccccgctg cggcgtgcct
  86041	gacctgcccg atgggccgag tgcccgcaac cgacagaagc ggttcgtgct gtcgggcggg
  86101	cgctgggaga agacggacct cacctacagg tagggccagt ggccacgagc tggcctttga
  86161	tctccacctg ctgtctgaga cacgctggag ctggggggag ggcagatccc tatggccaac
  86221	aggctggagt gtcccccaac tcccgtgccc actgctcaac accccaaacc cacacttaga
  86281	tgcactccca tgccctccct tgggagcacg gtctccacac ccacctggcc accccacaca
  86341	cccgtggggc acggccgtta gtcacccacg caacctctgc gggcaccgtg ctgcgggcca
  86401	ggccctggga ctctcagtga gggaggcaga cacggcccct cctccggggg agcgaggtgc
  86461	tccccacgcc cggttcagct ctagcaccgc actcgggacc ctcacaggga gggacccact
  86521	ggggcaggcc aggtgacggc tcgggtgacc tcggcccctg gcgctgagac tacacttcct
  86581	gcagtgggcg gcgaagatgg gtgtggtgtc ccacgtcgtt gcagcgggga ctcctggggc
  86641	ctcggaagtg tcctgggcgg ggagcctggg gagcaggaag ggcaggtctt ggggtccaag
  86701	gcctccccac ggtcaggtct gggagggggc ctcggggctc ttgggtcctt tccgcccagt
  86761	gcagaccctc gcggccacct aagggcacac agaccacaca aagctgtgcc catgcagtgt
  86821	ggggagtggt gcgcaccctc agagcacact gggcccacat cacgcacgcc tgccccctca
  86881	ctgtgcatcc ggggaaactc ctggccccga cagccagcgg ggctgacgct accccgtgag
  86941	ccagacccag gcccccctca ccgcccctgt cctccccagg atcctccggt tcccatggca
  87001	gctgctgcgg gaacaggtgc ggcagacggt ggcggaggcc ctccaggtgt ggagcgatgt
  87061	cacaccgctc accttcaccg aggtgcacga gggccgcgcc gacatcgtga tcgacttcac
  87121	caggtgagcg ggggcctgag ggcaccccca ccctgggaag gaaacccatc tgccggcagc
  87181	cactgactct gcccctaccc accccccgac aggtactggc acggggacaa tctgcccttt
  87241	gatggacctg ggggcatcct ggcccacgcc ttcttcccca agacccaccg agaaggggat
  87301	gtccacttcg actatgatga gacctggacc atcggggaca accagggtag gggctggggc
  87361	cccactttcc ggaggggccc tgtcgaggcc ccggagccgg gcccgggctc tgcgtccgct
  87421	ggggagctcg cgcattgccg ggctgtctcc ctcttccagg cacggatctc ctgcaggtgg
  87481	cggcacacga gtttggccac gtgctcgggc tgcagcacac gacagctgcg aaggccctga
  87541	tgtccccctt ctacaccttc cgctacccac tgagcctcag cccagacgac cgcaggggca
  87601	tccagcagct gtacggccgg cctcagctag ctcccacgtc caggcctccg gacctgggcc
  87661	ctggcaccgg ggcggacacc aacgagatcg cgccgctgga ggtgaggccc tgctccccct
  87721	gcccacggct gcctctgcag ctccaacatg ggctcctcct aacccttcgc tctcacccca
  87781	gccggacgcc ccaccggatg cctgccaggt ctcctttgac gcagccgcca ccatccgtgg
  87841	cgagctcttc ttcttcaagg caggctttgt gtggcggctg cgcgggggcc ggctgcagcc
  87901	tggctaccct gcgctggcct ctcgccactg gcaggggctg cccagccctg tggatgcagc
  87961	cttcgaggac gcccagggcc acatctggtt cttccaaggt gagtgggagc cgggtcacac
  88021	tcaggagact gcagggagcc aggaacgtca tggccaaggg tagggacaga cagacgtgat
  88081	gagcagatgg acagacggag ggggtcccgg agttttgggg cccaggaaga gcgtgactca
  88141	ctcctctggg cacagctggg aggcttcctg gaggaggcgg ttctcgaagc gggagtagga
  88201	taaaaggtat tgcaccccat gaagcacgtg tgatccttgc ccctagagac aaggctctgg
  88261	ggctcagagg tggtgaagtg acccacatga gggcacagct tggagaatgt cgggagggat
  88321	gtgagctcag tgtgccagag atgggagcct ggagcatgcc aaggggcagg gcctgctgcc
  88381	tgagagctgg cactggggtg ggcagccaag tgcagggatg gagcgggcgc ccaggtggcc
  88441	tctttgctgc tcagaacgac ctttcccatg tatacctccc agcgccgctg gcattgccca
  88501	gtgtccttct tgggggcagg agtaccaagc aggcattatt actggccttt tgtgttttat
  88561	ggacaacgaa actgaggctg ggaaggtccg aggtggtgtt ggtggcggaa ggtggccgct
  88621	gggcagccct gttgcagcac acacccccca cccaccgttt ctccaacagg agctcagtac
  88681	tgggtgtatg acggtgagaa gccggtcctg ggccccgcgc ccctctccga gctgggcctg
  88741	caggggtccc cgatccatgc cgccctggtg tggggctccg agaagaacaa gatctacttc
  88801	ttccgaagtg gggactactg gcgcttccag cccagcgccc gccgcgtgga cagccctgtg
  88861	ccgcgccggg tcaccgactg gcgaggggtg ccctcggaga tcgacgcggc cttccaggat
  88921	gctgaaggtg tgcagggggc aggccctctg cccagccccc tcccattccg cccctcctcc
  88981	tgccaaggac tgtgctaact ccctgtgctc catctttgtg gctgtgggca ccaggcacgg
  89041	catggagact gaggcccgtg cccaggtccc ttggatgtgg ctagtgaaat cagtccgagg
  89101	ctccagcctc tgtcaggctg ggtggcagct cagaccagac cctgagggca ggcagaaggg
  89161	ctcgcccaag ggtagaaaga ccctggggct tccttggtgg ctcagacagt aaagcgtctg
  89221	cctgcaatgc gggagacctg gattcgatcc ctgggtcagg gagatcccct ggagaaggaa
  89281	atggcaatgc cctccggtac tgttgcctgg aaaattccat ggacagagca gcctggaagc
  89341	tccatggggt cgcgaagagt cagacacaat ggagcgactt cactgtctta agggccacct
  89401	gaggtcctca ggtttcaagg aacccagcag tggccaaggc ctgtgcccat ccctctgtcc
  89461	acttaccagg ccctgaccct cctgtctcct caggcttcgc ctacttcctg cgtggccgcc
  89521	tctactggaa gtttgacccc gtgaaggtga aagccctgga gggcttcccc cggctcgtgg
  89581	gccccgactt cttcagctgt actgaggctg ccaacacttt ccgctgatca ccgcctggct
  89641	gtcctcaggc cctgacacct ccacacagga gaccgtggcc gtgcctgtgg ctgtaggtac
  89701	caggcagggc acggagtcgc ggctgctatg ggggcaaggc agggcgctgc caccaggact
  89761	gcagggaggg ccacgcgggt cgtggccact gccagcgact gtctgagact gggcaggggg
  89821	gctctggcat ggaggctgag ggtggtcttg ggctggctcc acgcagcctg tgcaggtcac
  89881	atggaaccca gctgcccatg gtctccatcc acacccctca gggtcgggcc tcagcagggc
  89941	tgggggagct ggagccctca ccgtcctcgc tgtggggtcc catagggggc tggcacgtgg
  90001	gtgtcagggt cctgcgcctc ctgcctccca caggggttgg ctctgcgtag gtgctgcctt
  90061	ccagtttggt ggttctggag acctattccc caagatcctg gccaaaaggc caggtcagct
  90121	ggtgggggtg cttcctgcca gagaccctgc accctggggg ccccagcata cctcagtcct
  90181	atcacgggtc agatcctcca aagccatgta aatgtgtaca gtgtgtataa agctgttttg
  90241	tttttcattt tttaaccgac tgtcattaaa cacggtcgtt ttctacctgc ctgctggggt
  90301	gtctctgtga gtgcaaggcc agtatagggt ggaactggac cagggagttg ggaggcttgg
  90361	ctggggaccc gctcagtccc ctggtcctca gggctgggtg ttggttcagg gctccccctg
  90421	ctccatctca tcctgcttga atgcctacag tggcttcaca gtctgctccc catctcccca
  90481	gcggcctctc agaccgtcgt ccaccaagtg ctgctcacgt tttccgatcc agccactgtc
  90541	aggacacaga accgaactca aggttactgt ggctgactcc tcactctctg gggtctactt
  90601	gcctgccacc ctcagagagc caaggatccg cctgtgatgc aggagtgagt gaagtcgctc
  90661	agccgagtcc gactctttgc aaccccatag gactgtagcc taccaggctc ctctgtctat
  90721	gggatttttc aggcaagagt gctggagtgg gttgccattt ccttctccag gggatcttcc
  90781	caaccctggt ctcccgcata gcaggcagac tctttactgt ctgagccacc aggcaatgca
  90841	ggagacctag gttcagtctc tgggtgggga agatcccctg gagaagggaa tgacaacctg
  90901	cttcagtatt cttgattggg gaatcccatg gacaaaggag cctggaggcc tacagcccat
  90961	agggtgcaaa gagacacgac tgagcaagtc acacacacag agccctacgt ggatgctcat
  91021	agcggcacct catagctgcc atgtatcagg tgttggcatg ggcagccatc agcagggggc
  91081	catttctgac ccactgcctt gttccaccgg atacacgggt gccttcctgt gtgtcgggcc
  91141	cactcggctg tcagcgccca agggcagggc tgtcgggagg cacagggcac agagttaagg
  91201	aggggatggg gacgttagct cctccccagc tctcagcgga tgcagcaggc aaaacaaacg
  91261	ctaggaatcc tgccaaaccc ggtagtctct gcccatgctc gccccatccc cagagccaca
  91321	agaacgggag ctggggggtg gcccggagct gggatactgg tccctgggcc cgcccatgtg
  91381	ctcggccgca cagcgtcctc cgggcgggga aactgaggca cgggcgcctc cggcttcctc
  91441	cccgccttcc gggcctcgcc tcgttcctcc tcaccagggc agtattccag ccccggctgt
  91501	gagacggaga agggcgccgt tcgagtcagg gccgcggctg ttatttctgc cggtgagcgg
  91561	ccttccctgg tacctccact tgagaggcgg ccgggaaggc cgagaaacgg gccgaggctc
  91621	ctttaagggg cccgtggggg cgcgcccggc ccttttgtcc gggtggcggc ggcggcgacg
  91681	cgcgcgtcag cgtcaacgcc cgcgcctgcg cactgagggc ggcctgcttg tcgtctgcgg
  91741	cggcggcggc ggcggcggcg gaggaggcga accccatctg gcttggcaag agactgagnn
  91801	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  91861	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnct gcaggtgccg gcggtgacgc
  91921	ggacgtacac cgcggcctgc gtcctcacca ccgccgccgt ggtaaccgcc cccgggggtt
  91981	gccaaggtta cgattggacc ctccccgccc cgaccctgct cccctagggt gggtgggtcg
  92041	gggggcagtt tctaagatct cctggttccg cagcagctgg aactcctcag tcccttccag
  92101	ctctacttca acccgcacct cgtgttccgg aagttccagg tgaggccgcc ccgccccttg
  92161	cacttgctgg cccaacccct cccgcccagc gctggcctga ccgcccccca ccccgcccac
  92221	cccacgcagg tttggaggct catcaccaac ttcctcttct tcgggcccct gggattcagc
  92281	ttcttcttca acatgctctt cgtgtatcct gcgccgtggt ggaagcggga ggagggcggg
  92341	gcgggggacc gggcgggagg cagcgggccc cgggaagctg agaccctcca aggggcacgc
  92401	ttcctatacc aaagccgcag gttccgctac tgccgcatgc tggaggaggg ctccttccgc
  92461	ggccgcacgg ccgacttcgt cttcatgttt ctcttcgggg gcgtcctgat gactgtatcc
  92521	ttcccgggct cggggaccta tgggtccggg cctctgctgg ccctgaggcc ctgcttgagc
  92581	gcatgccaca gagggagagt tgcgaccccg agctgagggt gtttttgagc gtacatcacg
  92641	tgctcagctg caggtgcccc tgtcgaactc cagggctaca cccaaaatac cacagggcag
  92701	ggtgcccagg ggctgagtcc tgaatgcagg tagccaggag gatctagggc tgggcccggg
  92761	ggctggggtg aagtggagag gcagggccga tcagggggcc cctggaggcc accgtttggt
  92821	cttagagtgg gaagcgaaac caacctgctt gagggtttca ggggtttagg aagtcagagg
  92881	ggccctgggc agggcacaag accttgactc tggcccagct actggggctc ctgggtagcc
  92941	tcttcttcct gggccaggcc ctcacggcca tgctggtgta cgtgtggagc cgccgcagcc
  93001	ctggggtgag ggtcaacttc tttggcctcc tcaccttcca ggcgccgttc ctgccctggg
  93061	cgctcatggg cttttcaatg ctgctgggca actccatcct ggtggacctg ctgggtgagc
  93121	ctgctgtcca gggagcctgc cccaagctgg gtgtgctggg ccagagccct ggtcctctcc
  93181	ccgcccccac ccctcttccc cactcctggc gcccccatcc ttccagcccc tccaacaagt
  93241	cagcctatag gttttactta ttcgagcctg acccatttgc tgacgcttgt gtggggcccg
  93301	acccggtagg gatgggtggc tcagggtgcc tgctcacagc tccacttctt ctgacgtcct
  93361	caggcctgac ctcctcccag gttctgccta ctctgggcca agcctggccc cacgctgggc
  93421	tggctggccg tgcagggcat cagaccccca tgctttgggg gcttcagggc tgtggagggt
  93481	ggcctcggca ttggcgcctc tcccacaggg attgcggtgg gccacgtcta ctacttcctg
  93541	gaggacgtct tccccaacca gcctggaggc aagaggctgc tgctgacccc cagcttcctg
  93601	tgagtgctga cagccttccc cacccccttc cccagatggc tctctacccc atgagggggg
  93661	gggaccctgc cagctgccgc tcagcgtggg ctcctcccca caggaaactg ctactggatg
  93721	ccccagagga ggaccccaat tacctgcccc tccccgagga gcagccagga cccctgcagc
  93781	agtgaggacg acctcaccca gagccgggtc ccccaccccc acccctggcc tgcaacgcag
  93841	ctccctgtcc tggaggccgg gcctgggccc agggcccccg ccctgaataa acaagtgacc
  93901	tgcagcctgt tcgccacagc actggctctc ctgccgcggc cagcctctcc acgcggggca
  93961	ggtgctgctg gccgagagcc agggccacca agcctgacgt gctctccgac ccagaacatt
  94021	ggcacagctg gaggcccaga gagggtccag aacctgccca ctcgccagca gaactctgag
  94081	cacagagggc agccctgctg gggttctcat ccctgccctg cctgtgccgt aattcagctt
  94141	ccactgatgg ggctcacatc tcaggggcgg ggctgggact gggatgctgg gttgtgctga
  94201	gctttggccg tgggggccct cctgtcccga actagcaacc cccaagggga cctctgcttc
  94261	atttcccagc caggccactg aaggacgggc caggtgcaga agagggccag gccctttctg
  94321	tgactccgaa gcctcaagtg tcagtgtttg cagagtccag tggctgaggc agaggcctct
  94381	gggaagctct gcccctgccg tttgcagctg aggccggcag gagcctcacc tggtccccag
  94441	ctcacgggca ttggaggacc agtccgcacg gtggtttact cctgggtcgg caccagccgc
  94501	cgccggctgt ccctttcaca gaggataaaa gtactcgctc tggagttgga ctttaatgtt
  94561	gtcatgaaac ctctggccca gcagcgggct ccgcagtggg tggcaggtga aggcccctcc
  94621	ccgggcctct ccaggcaggt gccgcctggc cagcagggaa ggcaggcagt gtcatccccc
  94681	actggctctg gggctcaggc tacctcctgc tgtggccgga acatctcccc cagtggtgga
  94741	gcccagtgtc cgtgaggcca gctgggcctg aaaccttcct ctctgaagcc ccgctgtccc
  94801	cttgccctgt atggagggca gaggctggag cgcaagttcc taggatgtgc ttgcgagacc
  94861	cccgagccca ggggcgaggc ccatctcagc ccacccccga actggaaacc cttggagctc
  94921	tgcccctcgt ggtgtgaggc ccctgctatg cgaccctcag ccctgccagc aacggaaggt
  94981	gcagggcccg ggcccacggg cttaacgcaa ctgggcctgg gtcacctgcg gggcctggtc
  95041	ccaggaggaa gacccaggtg ccaccctcct gggtgccacg tccaggtcac gtggggaccc
  95101	gtccatgtca cagaagatgc agggtcaccc ggtgagctgg cgccgggccc tgccagagca
  95161	ccagccgcgg gtggaggtgg gccccagctc tcctgtcagg cacgtggtgc tgggaggtgc
  95221	ggccggagca gtgcccacca gctgcagcag gacaggtggg cacaggccca ccagcagtgc
  95281	ccgcacggga tgggcccctg caagggccag agaagccacg ctcctggctg ggggctgggc
  95341	tgggactgac aggtggccct gccctctgcg ccccactact tcccagccac ccgggactcc
  95401	aaggacttgc tgagctgggc aggtgggacg ccgaggggag tcaaactgct cgtgggggca
  95461	ggaggggcgg tccacagggc tgagccctga gctgaaccct ggccctgctc gtggttgtgg
  95521	gggtgggggg gtccagtggc gccctagccc tgctgaggcc cagctgggac gtgcgcgccg
  95581	gagggcgagg ggccagccca tgccatgctg tcccccgttc tcagctccat gctaccactt
  95641	tgaagaaaca gaacctgttg cctttttatt tagaaagtgt tgcttgccct gcctggggct
  95701	tctatacaaa aaacaaacac agctcaacgt ggcctctcct gaccagagac gggcggtggg
  95761	gactggggct cagcagacgg aatgtgtccc cggcggcggg agaccaggag gcccctggcc
  95821	cgctcctcag gacggctggg ctgtccccac ctggtcccct ccgagccaga agatggagga
  95881	gaggtgggct gatctccaga tgctccctgg gagccaagcg ccacggggtg gtcaccaggc
  95941	cggggccgtg ttggccagac gcctcatccg cctgtgggag ggggagggca gcaacccccg
  96001	gatctctcag gcaaccgagt gaggaggcag gagcccccag cccctccctc ggccgctctg
  96061	ctgcgtgggg ccctgaagtc gtcctctgtc tcgcccccct ccccagggag agtgagcctg
  96121	ttctgggctg tggtcagacc tgcccgaggg ccagcctcgc ccggggccct gtcctgcctg
  96181	gaaggggctg gggcagcacc ttgtgttccg gtcctggtcc cggatcttct tctccatctc
  96241	tgcatccgtc agggtctcca gcagcgggca ccactggtca gcgtcgcctg tgttccggat
  96301	ggcaatctcc accgtgggca gggggttctc actgtggagg acgagagagg tagacggctc
  96361	acagagcagc tgcaggagag gcccctagaa agcagtgtcc accccgctgc gggcagacag
  96421	gacatggagc ctggtttctg cacccggctc ccgacacagg gcggccgggc acgctgccaa
  96481	catggcatct ccgggtctgc atgtggggag gggtccacag gacagtgctg caggtccagc
  96541	cattcccagt ggacttgctg ggaggaggag ggccgtccgc cccgctcagt gtccaggaga
  96601	aaggagagca aaggagtcca tccacccagg agtggagtcc cagggcccct gccctgacca
  96661	gcctgcaggg ggcccctcgg cccacatcac aggggcccag aatccataag ccctgactgc
  96721	tccaccccgg ggcccctcaa agacgcgcct agactccgtc cgagggccac ctgcacaccc
  96781	tctggcgaag tggactcagg gctgggggtc agcctcggtg aggccgcaaa ggctggggac
  96841	tcctggccga gctgctgcct ctgccaggag ccaggcccag cctgccggcg agcctcagcc
  96901	acgccctcac ccaccctgcc cgcggcgcca cgctggcctc cgggtcctct cctctggcct
  96961	cctgctgggc cactggtgct cagccccagc agtcggcctg ccaggagccc tgcagagtca
  97021	gcccccagag ggaggagggg gcccggggga acagcacagg aacaaacaga cccctggcct
  97081	tagttttagc tcctcatctg gaaaatgggg acagtgtcct tgctgcgagg ggtttcagag
  97141	gaccactgcc atgcaacacc cagcacacac ccactgcgtg ggggctcggg cccgagccgg
  97201	tgcccccgag tcccaggctg gtggctgggc cgccccagcc accctgccga cagctgcttc
  97261	ccagccgggc ggtgctgcgg cagtccagaa gccagcactg cagacccaaa tgtcactcct
  97321	cacgttgcgg gctcccagct gccttccttg ggggcagcag acacgaaagt caccaagccc
  97381	acgccgacgg gagcaaacac gtcttcctct taaacaagtg cgggtcccgg aggccctgtg
  97441	tttacctccc tgtggctccg ggaagattgc atcccagggg gttgttctaa accaagggct
  97501	gctcgggcca ggcctggaag gaggggcctg gagccaggag cccaccctta cgggcattcg
  97561	gcttcctggg tctcaaggcc ggctgggacc ctgcattccc accacccgcc aggtgcaagc
  97621	agggaggccg tgtcggagga ggcagagggc ctggagggtc gtcttcgacg tgacctcact
  97681	tttacaacct cacaggtgcg gcaggccagc tgggaggcat ggctgtgccc tcctggtaga
  97741	tgagaacaag actgcaggga gtgatccccc tgaacttccc caaccaggag gagacaaaac
  97801	tcggtgtcgc cctcctgctt aagatcaact gactctggac aaggggccca gcccacccga
  97861	tggggaaagg gcagtccttc caacaagcgg tgctgggacg ggacccggca ggccatggtt
  97921	tctcagctat gacaccagca gcacaagcac cccgagaaaa acagctaagc tgggcactgt
  97981	cacacaagtg aactccaaac ccaagaaaac cacaaaaagc ctgcggatct tcagatatgt
  98041	gggaagggac ctgtatctgg aatgtataac gaactcctga aaagtgaaag tgttagtcac
  98101	tcagtctgtt cagctctttg caaccccatg gacggtagcc tgccaggctc ctctgcccat
  98161	gggattctct aggcaagaat actggagtgg gttgccatgc cttcctccag gggatcttcc
  98221	caacccaggg attgaacctg tgtctctctt gcactggcag gcgggttctt taccagtagc
  98281	gccacctgag tagaaacact ccaggtgccc tgagtgtcag agcaggaggg actcggccca
  98341	ggcctgtgag gggaccctct ccgagtcccc tgctgcacag cagtgagagg tgcgttctga
  98401	gtcagcctcc agggatgagg gacttggtgt cgacatcact cccaggacct caggatctgc
  98461	tctgggaagc gaggctcccc aggctggccc caggcccgct ggcctcagct cgtgagccgt
  98521	gcgtggacag gtgccatgag caggcctccc acgggactcg gggcgcggcc tggaccccgg
  98581	ggctgccagt ggtcgcgggg ggccccgtgt ggcggctgtt ccctctcttg ctccgagtcc
  98641	taggaacatg gtgggcgctg cctcctgggg tttctggaga agcagctgag atgcaaacag
  98701	ccccacgcgc tccctcagct gttccctgtc acgggtggcc ccttggtgac ggcctccatg
  98761	cagggacggt gacagctcga gcagccgcgt aaaaccacac ggggacggtg gcagctcgag
  98821	cagccgcgta aagcctgaca tccaatttgg aagcctcccg cagtggaaga ggggcccggg
  98881	gacggggctg cccggggcga gctccaccgg gtcgggggtc acgaggagcc cacccgcgtc
  98941	cccgccacca gcacctggga ccagataccc tccccgctct gagggcggcc tgaacgccgc
  99001	cccctcccac gggggcgccc accgcctgct cgtggactga acaagaggcg gcagtggcct
  99061	ccagaccccc tcgggggagg gcagacctgt ccgagactga gcacaagtcc agggaatgag
  99121	caagggtctc agtaatgtcc ccaccgggac gggacgggag gaggcgacag aggccgctga
  99181	ggtgcggggc agccctcagt agctggcatc aaggccccag gcagtcccgg ggcatccccg
  99241	cagggggcgg gggcgaccac cggcccgagc ccaggcagtc ccggggcatc cctgcagcgg
  99301	gcgggggcga ccaccggccc gagccctacc tgaaggcgta ggtcttctga tgccagctca
  99361	gctgtccccg gatgctgtag gcgatggtgg tgacgaactc cccgcccagc cccagctcgg
  99421	agcacagctt cagagcgaac ttctcgggcg agttctcctt ctccgacatg tcccactcga
  99481	actggtccac caaggagatg ttccccacgt ggatgttcag ctggcccggg agcacagaca
  99541	tgagccagag cggccccctc tggggccagg ccgcaccctc accacccctt ctccccggaa
  99601	catccccgcc tcgttcttgg ccgcgcccct gtgctgctac ttggggtaag gaaaacaacc
  99661	cccatctctc tgaaaagggt taactagcga ggaagatgcg ctggtaactg gaaaactccc
  99721	tacaaagaaa gcttggatct gatggcttca ctggtgaatt ccaccaaaca tttcaagcac
  99781	taacaccaat ccttatcaaa tcctgccaaa aaactgaaaa ggaaggaaca catcataact
  99841	ccctgccttg ataccaaagc cagacaaaga tactacgaga aaggaaaggt gcagaccggc
  99901	acttactgtg gacattgatg tgaaacctca gcagacacga gcaaaactac attcaccagc
  99961	acgtcagaag aatcacacac cgttataaat gatgggatga tgacacaacc acattataaa
  100021	cggtggggct tactctggtg atgtaaggac ggctcagtaa gaaaaccggt caatgccatg
  100081	aaccacttga acagagtgaa ggacaaaaac cacacagtca tcttgataat tggaggaaaa
  100141	tcattagaca aacttcaacg tgctttcacg ataaaagcac tcagtaaact aagatcagat
  100201	ggaaaccaca tcaacaagat taattcagtc aaaaaattca ctgcaagtat cacccacaat
  100261	ggcagaagac tggtaacttt tcctctaaga tcaggaacga gccaaagata cccagtcttg
  100321	ccacttttgt tcaatatagc gttggaattt ctactcagtg cagtgcagtc gctcagtcgt
  100381	gtccgactct tttcgacccc atggatcaca gcacgccagg cctccctgtc catcaccaac
  100441	tcccggagtt cacccaaact catgtgcact gagtcagtga tgccatccag ccatctcatc
  100501	ctctgtcgtc cccttctcct cctgcctcca atcccttcca gcagttaggc aagaaaaata
  100561	aatcaaaggt atccacctgg aatggaagaa gtaaaactat ctctggtccg agatgttaca
  100621	atcttatatg cagagtttaa gatgctaaca aaatactatt agaactaatg aatgaattca
  100681	gcaaggtacc aggatacaaa gtcaacgtgc aaaaatcagc cgcatttcta catgctaaca
  100741	ctgcacaatc tgaagaagaa aggatgaaca aattacaata acataaaaaa gaataaaatc
  100801	cttagaaatt aacttgatca aagagatgta caatgaacaa tataaaacat actgaaagaa
  100861	attgaagata taaataaatg gaaaaacatc ctatgtccat ggattggaag acttaaaatt
  100921	attaagctgt caaggctatg gtttttccag tggtcatgta tggatgtgag agttggacta
  100981	taaagaaagc tgagcaccga agaagtgatg cttttgaact gtggtgttgg agaagactct
  101041	tgagaggtcc ttggactgca aggagatcca accagtccat cctaaaggag atcagtcctg
  101101	ggtgttcatt ggaaggactg atgttaaagc tgaaactcca atactttggc cacctgatgc
  101161	gaagagctga ctcatttgaa aagaccctga tgctgggtaa gattgagggc gggaggggaa
  101221	ggggacaaca gaggatgaga tggttggatg gcatcaccga ctcaatggac atgggtttgg
  101281	gtggactctg gaagttggtg atggacaggg aggcctggcg tgctgcggtt catggggttg
  101341	tgaggagtcg gacacgactg agcgactgaa ctgaactgaa catgaatacc caaagcaatc
  101401	tacaaagcca aatgtaatcc ctatcaaaat cccaatagca tttctgcaga aacaggaaaa
  101461	aaaatcttaa aattcatatg gaatctaagg aaaagcaaag gatgtctggt caaaacaatg
  101521	acgaaaagaa caacaaagct ggaagactca cacttcctga tttcagaact tactgcaaag
  101581	atacaataat gaaaacactg tgggactaac gtaaaagcag acacgtgggc caacgggaca
  101641	gcccagaaat aaactctcaa ataagcagtc aaatgatttt caacagagat gccaagacca
  101701	ctcagtgaag gaaagtgttt gcaaccaacg gttttgggaa aaaagaaccc acatgcgaaa
  101761	gaatgaagtg ggacccttac ccagccccat ctacagaaat caactcaaaa cagacagaac
  101821	atatggctca agccataaaa cgctcagaaa aacagagcaa agctttatga tgttggattt
  101881	ggcggtgatt tctcagatat gacgtcaaag gcataggtga taagcgaaaa aataaactgg
  101941	acttcaccaa aatacaacac ttctatgcat ccaaggacac taccgacagc ataacaaggc
  102001	agcccaggga aaggaggaaa catccgcaaa tcacagcatc tgggaacaga ccgctgcctg
  102061	tgagatacag ggaaccgata aaaacaagaa aacagcaaaa cccggactca aaaatgggaa
  102121	ggactccagc agacacagga gacagacaag ccgccagcag gtcactaatc agcaagcaag
  102181	gcccgcaaag gcccgtatcc aaggctgtgg tttttccagt ggtcatgtag gaaagagagc
  102241	tggatcgtaa gaaagctgag cgctgaagaa ttgattgaac tgtggtgttg gagaagactc
  102301	ttgagagtcc cttggactgc aagatcaaac cagtccattc tgaaggagat cagtcccgaa
  102361	tagtcactga aggactgatg ctgtagctcc aatactttgg ccacctgatt cgaagaactg
  102421	actcattggc aaagaccctg atgctgggaa agattgaagg caggaggaga aggggacgac
  102481	agaggatgag atggttggat ggcatcactg actccatgga catgagcttg ggcaagctcc
  102541	gggagagagt gaaggacagg gaagcctggc gtgctgcagc ccgtgggtcc caaatctttg
  102601	gaccaagcga ctgaacaata acaaatcaac agggaaatgc aaatcaaaac cacagtgaga
  102661	tactgtccac caccaggcag gcgttcttca gcggggttcg gggcaggtgg tgccctcttc
  102721	tctcgtaacg cccccaggac cgcgggggct gctgagacag catggggtgt gcttggccta
  102781	gcctgcccat gacaagagtg gcagtgtgct cgcctcactg cgcccttccc tgctctgccc
  102841	accagctggg ccacccctgg gaccacccag cttccgctcc gtggacggca aggccgcagc
  102901	agcgcccgga cacgcccaga acgtggtgcc ctcctcagaa gtcggcctgt gcccttcctg
  102961	ggacaagccg cccaagagac agtcttccag agccctgccc cacaacacgg accccagaca
  103021	ggctcctgtg gaggcctcca cgcacctccg cacctcgcaa gccccgagga caaggcaggc
  103081	ccgctgcggg tgaggagccg cctaccttga taatgacgcg ctggtctgac tggtcttcca
  103141	ggatgctgtc cgtggggtag gactcgatct gctgtctgat ggcagaggca atggctggca
  103201	cgaatgtcag tgggttcaga tccaggtcgt cacagagaat ctctgagaac atctccgggg
  103261	tcatcagctt ctctgaaacg atgacggagc gggggaaccc ccagtggacc acagggccta
  103321	cggtcagcgt gctcagcccc ggcctccccc agccttgcct cctctgccac cgcccccccg
  103381	ggtgacgaca ggaccccctg gcagcacgca gacagagctg agtgcacgcc agccagggcg
  103441	gcggacggac cattcatgtt ccaggtaaag gcatcccgca gcttctgccc gtcaatctcc
  103501	atgtccagtc ggatggggac cagcacctcg ggctgggacg cgttctcgtg gatcacggct
  103561	gggtcgtggt cgtcgaagct ggaaggggag cggccgcgtg ctcagcaaag cgggctgggc
  103621	ccctgtgccc agggcctccc tctctgcacc actggtcgct gagacctgcc cagagaggac
  103681	ctgtccacta cgggccgggc cggcagaaac agggctggcg ggggtccacg cggggcggga
  103741	ggggagctgc cgactcggca gcgggacaag ctcagaggtt ccctgcagga agagaggttt
  103801	aagccccaga gcaggcagga ttctcccagc agctgtgggg aagaaagggt atgtccagaa
  103861	gaagaaaccc tggaacaaag gccgaggggc aggagggttg aggagctgct tggagagcag
  103921	tgaagggggg ctgggcggct ggggggtgct ggggagcctc ggtggccaag cacccagggc
  103981	tccccacctg cagcctggac cccgagggag ccccagagga cggagagcaa ggcagctccg
  104041	cactcacacc tgccctttag gatggggaag agggaagaga cgggggctgc ggggggcaag
  104101	gaaaccaggc acgccccgct tagacccggg ggcgagaacc actttccaag aacgcagggg
  104161	cgccaatgat gaacaatggg tagcagcccg caggcgggag gcccggtggc cgaggcccct
  104221	caccagagcg ggaaggtccg cttcttgtcg cggcccatgc ggttcctgtt gatggtggtg
  104281	gagcagggca cggcgtccag gtggtgcgag ctgttgggca gggtgggcac ccactggctg
  104341	ttcctcttgg ccttctgttc cctgggagac acagacgccc gtccgctcag cctatgggcc
  104401	aaaagccgcc ccccagccgc caggttgtgg ccagtggacg cccgccatgc ccctctgggc
  104461	ccaggccccc atggggacct ctgtgcgccc agctccgcgg tggttattcc ccaggctcca
  104521	agcggcacct gctcggggtc accagtttta ggggaggagg agagggcagg ggccccagcc
  104581	cagtctgtga gctgtcaccc ccaggctcca agcggcacct gctcggggtc accagtttta
  104641	ggggaggagg agagggcagg ggccccagcc cagtctgtga gctgtcaccc ccaggctcca
  104701	agcggcacct gctcggggtc accagtttta ggggaggagg agagggcagg ggccccagcc
  104761	cagtctgtga gctgtcaccc ccaggctcca agcggcacct gctcggggtc accagtttta
  104821	ggggaggagg agagggcagg ggccccagcc cagtctgtga gctgtcaccc gtgctatgtg
  104881	ctgggctggg cactcaggaa agagggtcag ggttcacggg ggggtggcgc gcagatttcc
  104941	aggagagccc cgagggcagc agagaggagg ctcaggtcaa tggttgggca gggggccagg
  105001	gctggagaca cagagagggt cccgattcgg gggggtgccc tcagcaggtg gctgggagtc
  105061	cctgggggtt tgcacacttt cgatcaggct gttatttcag acgcttggtc cagcctgaga
  105121	caggtaatgc ctctggcctc cgggccttca gggatggaaa gatactctag aaagcgggac
  105181	tcaaagtaac tcaaggaact cgcgtcccac agtggggagc ccttctctcc aatttacatg
  105241	gggcgtttac tacgaggaaa ataccgaagg ccgttttgag ctgaggctcc cgggccgggc
  105301	tgtccgtttg tgagactgct cgtcacccct gggccacatc cctggtggcc aagggggcaa
  105361	tcagtgcggt gactgcacga cacacctctg cagccctgcc ccacagctgt caccatcggt
  105421	gacgtccacc ccctggagaa cctgaccact gcccggtttc ccgctaaaac agcgcccttc
  105481	caggatgggg ggcagaggga gaggccttgg ccttttcact cctcttctgc agcgggggcc
  105541	cctcgcaccc cagtgcccgg gcccaggagc gccccttggg gtggggcagg gagggatcca
  105601	cacaccaagg ggagccagga cccccccaaa tctgctgccc tgccctgata cccgagacct
  105661	ggggaaacgg gggactgggg ctgatgcggg caggaccaag aactgaggcg gtgagacggg
  105721	gtccccacca caggccatct ggctggcagt ttctactccg ggcctgcagg ccaagaggga
  105781	aaaggtgccc cactcagatc aggcgcctcc cgtccccagg gagggcctac aaggtcagat
  105841	cctttgtaac ttccacgggc aaaactggct tgctgggcct gtgcgggccg catgggcgtg
  105901	gaccaccaca cctttcccca ctgagtctcc agccggagct gtcacccagg tccccccagg
  105961	ccagccccac cccgccacct tgcagtagcc tctcgtatcc aggccgaggc tgcccggtcg
  106021	acccctcctg cctgatggcc tcaagtggac aatgcgagtc acgttgcagc acgtgagtgg
  106081	gacgggcagc gccacgcggg gtccgggcat ccgagtccca ccactcagcc tcccttccgc
  106141	tgcagagagg tctgtccaag agccctgggg gccatccagc ccctgtccga cctggccggt
  106201	gtggaagagg gggtgtgcca cccctcctgg ggggctggct gggcgctggg caggcccctc
  106261	ctaagagtgg agcccactgg tggttttcct gcagccccac ctccacacag cagttctcac
  106321	tgcccagtaa caggaggcta ctggcctagc tctctccctc gtgtgatgga ctcaaccagg
  106381	agcgttcacg gccccacaca gggttctcgg ctgctgcatg aggatctcaa agccccatcc
  106441	acgtgcatgt aatctcctcc ggtaacttct ctagggaagc ccggctatcc tgccatcctc
  106501	accgcaccac cagggcgaga aaagccatct ccagcgctca catccacaat gggccaggcc
  106561	gtgagcacac caccttcttc gggaggttgt gggggcgggn nnnnnnnnnn nnnnnnnnnn
  106621	nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
  106681	nnnnnnnnnn nnnnnnnnng cgcgcccccc ccccccgcgg cgccggcacc ccgggcggcg
  106741	gcccccggcg ctgggagcag gtgcggggcc gcggccgctc gtgagcctcc agcccggagg
  106801	acgggccccg ggggccggcc cggtgcccag gccctgggag ccccggaggc cagagtgcca
  106861	gagggccgga ggacccggga aggcccgaga gaggtgggaa gcacggggtt ccagccctag
  106921	gccatttcag ccccaaagcc atcggtgaaa ccattgctgg ccccagataa aagcgtcgcc
  106981	aactttttca ccccggcgga gactttagcg ggtagctgcc ccctaggggg aatggaaaaa
  107041	ccaggattta ccaggtgggt ggaggtcaca actgcccaga tcctgagaaa gaggggtcag
  107101	tggggcggga agattagtgg ggagaggagc tttcagaacc caagggaatg aaacgaggct
  107161	tgaggttggt tatccagcag ccgccccctg ccccgtgagt gagcgaaggc tgggcccctt
  107221	attgtcacat cttccagctc ttcgctagaa aacctagagt tttaaatact gtggcagctg
  107281	agtcaaacaa taaggaaaag cccgactctt tgagagccag gcacaaggcg tctgtgacag
  107341	ggtctccagg ctgcccattt gcagtctctg aaacggaggg tttttcgaga aggaggtctt
  107401	ggggtgcctg ccagaattgg aggggggggc gcgggaagtg aggacccaga agagagggct
  107461	tggcccgctg caaggaggtc actggacact ggagctgaag cgccagccga aactggaaac
  107521	tcgaaatctg tctccgtgcc agccacaagg cctatgattt tccttggcga cgttcagcat
  107581	cttaggagga gctggcgggg gaggcgggta gttcgtgggc ggttgcagca gggcaggaag
  107641	gtgaggaacc tgaggctggt cagagagctg gttggagtga tgcccatcgg tggacccgct
  107701	ggagaaggcc tgagtagaga aggtctaagc ttaacgggga aggggtgggc cagggtggaa
  107761	atggggtggg aagtttgagg agggggagca gtggagatgg gggttgtgag gaatgggagt
  107821	gagcttagac gtcttgagga tactgcagtt ctgtgctttt tttcacacct ggctgaaaat
  107881	tcactgaaaa caaaacaacc cttgctctgt gacagcctag aggggtggga gggaggctta
  107941	agagggaggg gacgtgcgtg tgcctatggg cgattcatgt gggtgtacgg cagaaagcaa
  108001	cacagtatgt aattaccctc caattaaaga tcaagtacaa cttaaaaacc ccaaacacaa
  108061	cattgtaagt cagctagact ccagtaaaca tttcagtaag aagattcaac tgggaatgag
  108121	ttccgccgtg actatcctga tgaatttccc gtgtcttctt gaggccattc ctctttgaac
  108181	ttccgtgttt ggggaagcgt gcctttgtat ggagtcctga ggagtaaatg agacgggctt
  108241	gtagaaggcc tagtagtgcc ttgcacgcgg cagatgctca ataacctcga gttgtcacca
  108301	ttatggtacc tcaagagtct ccttggagct tgcacggttt ctgaatgggg tcctgcgggg
  108361	ctcccttggg gctcccacat ggggttgggg ggctgagtgg ggtgtccccg ctccttgctt
  108421	gtcccctgtg gaacaccccc ttccacccga gcagctctgc ttttgtctct tgtgtttgtt
  108481	tatatctcct agattgttgt tcagtcgctc agtcgtgtcc aactctccga ccccatggac
  108541	tgcagcacac caggccttct gccttcacca tctcccggag cttgctcaaa ctcctgtcca
  108601	ttgagttgct gatgccgtcc aaccatctcg tcctctgtcg tccccttctc cttttgacct
  108661	cagtctttcc cagcatcagg gtcttttcca atgagtcagc tctttgactc aggtggccaa
  108721	gtattggagc ttcagcttca ttatcagtcc ttccaatgaa tattcagggt tgatttcttt
  108781	taggattgag tgacttgatc tccttgcagt ccaagggact ctcaagagtc ttcaacacca
  108841	cagttcaaaa gcatcagttc ttcggcactc agccttcttt atgatccaac gcccacatcg
  108901	gtacatgact actggaaaaa ctttggctca gagataattg acttgattga atacaaagtt
  108961	ctttggcaaa aaataaaagt gtggcaagca gtactgacac aaaagcaagt ggcttttcct
  109021	ccgttgagtc atttatttat tcagtgggtg tgtgcgtgta gagacggagc ggctgtgctg
  109081	ggagctgggg cttccacttc agaggagccc cggacctgcc ctcggggagt tcacaggcag
  109141	tgctgcgggg ggtcctgcca ggacgcctgc cctgcgagtg cccagtgctg tgatggatgc
  109201	gtgtcccgca tctgcggcca ctggggccac gtgcccgaga ttgtccgggt ctgagggtgc
  109261	agagaagagg aggcatttgg actgagtctg gaaaaatgag catgtggcca cgtgagaagc
  109321	cagtggtgag gggaccagtc aggcggagga aagagcggct catacgagtt gtggagctgg
  109381	aagcatgagg gtgtgtggaa gcagaggccg gggacagggc cgcagggccg gccatggagg
  109441	gcgtgggctg ctgcaggctc ctgagaaggg ggacgctgcc atcatgaccg ggtttaggtg
  109501	tttgaccctg gtgtccacgt agaggacaga tgtgtggggg gggagctgga gatgggcatc
  109561	catcgggagt cagcctggag agaggcagag accccgtcag tgggccctca ggacgtggat
  109621	ggggcggatg ttgggaagat ctgactcctg ggttccggct ggggctccgg gctggagggg
  109681	tgccgcccac cgagcacagg aggcaaacag atgccctctc ccagcaagac cccagcccca
  109741	gcaccctccg gggccggact ccgcccctct tccagaatgg ctcccttgct gtcctcgccc
  109801	atctttccgg tgccctgagc ctctagagtc tggacaccag cgtccgcctt gcgcttgttt
  109861	ctgggaagtc tctggcttgt ctctgactca cccaggaccg tcttcgaggg caaggttgtg
  109921	tccttggttc catctgcttt ggggtccggc tcctcgctgc ttgacctgct gatgtgacag
  109981	tgtctcttgt tttcttttca gaatccgaga gcagctgtgt gtgtcccaga cagacccagc
  110041	cgctgggatg acgggcccct ctgtggagat ccccccggcc gccaagctgg gtgaggcttt
  110101	cgtgtttgcc ggcgggctgg acatgcaggc agacctgttc gcggaggagg acctgggggc
  110161	cccctttctt caggggaggg ctctggagca gatggccgtc atctacaagg agatccctct
  110221	cggggagcaa ggcagggagc aggacgatta ccggggggac ttcgatctgt gctccagccc
  110281	tgttccgcct cagagcgtcc ccccgggaga cagggcccag gacgatgagc tgttcggccc
  110341	gaccttcctc cagaaaccag acccgactgc gtaccggatc acgggcagcg gggaagccgc
  110401	cgatccgcct gccagggagg cggtgggcag gggtgacttg gggctgcagg ggccgcccag
  110461	gaccgcgcag cccgccaagc cctacgcgtg tcgggagtgc ggcaaggcct tcagccagag
  110521	ctcgcacctg ctccggcacc tggtgattca caccggggag aagccgtatg agtgcggcga
  110581	gtgcggcaag gccttcagcc agagctcgca cctgctccgg caccaggcca tccacaccgg
  110641	ggagaagccg tacgagtgcg gcgagtgcgg caaggccttc cggcagagct cggccctggc
  110701	gcagcacgcg aagacgcaca gcgggaggcg gccgtacgtc tgccgcgagt gcggcaagga
  110761	cttcagccgc agctccagcc tgcgcaagca cgagcgcatc cacaccgggg agaagcccta
  110821	cgcgtgccag gagtgcggca aggccttcaa ccagagctcg ggcctgagcc agcaccgcaa
  110881	gatccactcg ctgcagaggc cgcacgcctg cgagctgtgc gggaaggcct tctgccaccg
  110941	ctcgcacctg ctgcggcacc agcgcgtcca cacgggcaag aagccgtacg cctgcgcgga
  111001	ctgcggcaag gccttcagcc agagctccaa cctcatcgag caccgcaaga cgcacacggg
  111061	cgagaggccc taccggtgcc acaagtgcgg caaggccttc agccagagct cggcgctcat
  111121	cgagcaccag cgcacccaca cgggcgagag gccttacgag tgcggccagt gcggcaaggc
  111181	cttccgccac agctcggcgc tcatccagca ccagcgcacg cacacgggcc gcaagcccta
  111241	cgtgtgcaac gagtgcggca aggccttccg ccaccgctcg gcgctcatcg agcactacaa
  111301	gacgcacacg cgcgagcggc cctacgagtg caaccgctgc ggcaaggcct tccggggcag
  111361	ctcgcacctc ctccgccacc agaaggtcca cgcggcggac aagctctagg gtccgcccgg
  111421	ggcgagggca cgccggccct ggcgcccccg gcccagcggg tggacctggg gggccagccg
  111481	gacggcggaa tcccggccgg ctcttctctg ccgtgacccc ggggggttgg ttttgccctc
  111541	cattcgcttt ttctaaagtg cagacgaata cacgtcagag ggacgaagtg gggttaagcc
  111601	cccgggagac gtccggcgag ctctaacgtc agacacttga agaagtgaag cggactcgca
  111661	gcccgtacag cccggggaag atgagtccaa agtcgagggt caccttggcc actgcagggt
  111721	cgctcggcgg tggggcggag cgggtgcagg agggctcctc ctgggcttgg ggtggcaggc
  111781	gaggaccccg cgcctctcag ccctcggcct gggttggctg agggcgggcc tggctgtagg
  111841	ccctccagcg gaggtggagg cgctgcccgg ctcagccagg cacaggaccc tgccacgagg
  111901	agtagccctc cgccagaccc ggcgtccagg ctggggcgcc tgcggggcct ccgttctgtg
  111961	gctgggcagc ctgcgccctg tccagggatg aaggggttcc ggtctgaagg gctgggttca
  112021	gggtccagct ctggcccctc ctgccttggt gtcctggagg aagccccaag gctccgtttc
  112081	cctctccagg aggtggggac gttgggaatg ccacattccc ctggggggtg tgtgtgtgtg
  112141	ttcaaggctc ccattcagac tgggactggg cactcacgag ctttggcaac tggcaactga
  112201	ggacggagac ccagggtgac accccacctc ctgctgcggc ccccccggca ggggagacac
  112261	aggcccgtct ggttcccaag atggcagggc ccctccccct ccagcttgtg ccctgggtgt
  112321	ggtgcctggg gctacagcga ccctttccgg ttccccgggc cagttcagct gggcatcctc
  112381	agggcggggc tctgagggtg ccatgtttcc agagctcctc ctcctcccac cagtagcagg
  112441	cgggcggcca gctcccaggc agccccctgg catcgcctag gtgcacacct gcccgctgtg
  112501	acccagcaag gcttgaaggt ggccatccca gttaagtccc ctgcccctgg cccaggaatg
  112561	ggctcgggca gggccgcatc tggctgcccc agaagcgtct gtccctggcc tctgggagtt
  112621	ggcggtggtc tctggtactg tccctcgcag ggccccttag cactgctcgg ggaggaggtg
  112681	ggctgaactg attttgaagt tttacatgtc tgcggccgca gtcctacgag cccgtcaggg
  112741	tcatgctggt tatttcagca gatggggctt ggctcggcag ctaggatggt cctgaataaa
  112801	aatgggaagg ccagagctgt tcctccatca gcaggcttgg cagctgggga cgttgaaagg
  112861	acaggtctgc tggtctgggg agaccagctc tgtgcagccc ctgctgtccg tgggggtact
  112921	aaaccagccc ctgtgtgcgc ccatctgagt ggcagcccgc ctggaggatc gcccatcact
  112981	tgtgagaatt gagagaatgc tgacaccccc gcttggtgca gggggacagg gccccctaag
  113041	atctacctcc ttgccccacc cccgggaccc cctcagcctt ggccaggact gtccttactg
  113101	ggcagggcag tcatccactt ccaacctttg ccgtctcctc cgcgcgctgt gctcccagcc
  113161	aaattgtttt atttttttcc aagcatcact ttgcacacgt caccactctc cttaaaacca
  113221	cccttccgga gtctcctgct cgtaaatcgc cggtttcagc caacctgggt cgccccccaa
  113281	gcccagcaag cctgctgagc cccgcgcctc ccagctactt cacgctcgcc tcaagcttct
  113341	aaacgcggac cttctccccc ccacccccat ccctttcttt tctgatttat gtaacacggc
  113401	aggtaagact cctctcctga agggttgaca gactcacaca aaaccgtggt cagaccaggc
  113461	aagtgctttt tttcagaagt gtgagcggaa cctagtcttc agctcatgct ctttccttgt
  113521	tttcttatgt gttctaagtc ctttgacttg ggctcccaga cagcgacgtt gtaagaggcc
  113581	gtcctggtag catttgaatt gtcctcgagt ttcgttgtcg gattttgttt tattgtctta
  113641	gttttccctt cttttagcag acgttgttga ctgtcgtaaa gctccagttc ttggttctgt
  113701	ttactaatca aattgttttg tcaaagtaca tgtattctgc tcttttcttt atcttttttg
  113761	ttgcttaata ttaacacttt acatttctaa gattaattat ttaggtaatt aataattttt
  113821	aacatttcta gtaaacgtgg gtacttgggt ctgtgtttgt tttcttgtag ttacagcttt
  113881	ttctgctcta tactgttgac gtctgggttt ttttttgctc ttaggaattt ccctttgacc
  113941	ccattattat tattttaatt agtatttttt aataattaaa aattagtgtt tttaaattaa
  114001	ccctaatcct aaccccagtg atgactgctt cagtcattgc tgttacttat tatgtgctgg
  114061	tgtcaggatt tttaagtgtc catagacatt ctctgagcct gaatatatta tcagttttat
  114121	acagcatttg tgtactctca agaaacgtgt tttcactctg tcagttcggt ttgttacctc
  114181	agtctttatg ttattttgct ccagtccgca cttgctctaa cttgtcttcc cttcgaggtg
  114241	tgaggacgcc tggcagccgg tgagcatgcc ggggtccggg gtcgtgggcc caggcgccca
  114301	gcaaagccct gtgggtgtgt gcacggctgg gctgctccgg gaggaagcct gtggccccac
  114361	ggtagttagg agcgctggtt tacctggtca caccacggtc tggttttgtg tgcttttccc
  114421	tgacgtgttt ctgttttgcc ttggtttcta ttctgtttta tgagtgccgt ttacgctttg
  114481	ttagtcatgc cgttatctcg atagacaggg tgtacgtgat caagtgatta ccgtatttgg
  114541	agcagatgtc tatttaacag agatgaactg agaacctgtg cctttgcatg ccctctttgc
  114601	ctcttttaat gcttctagct tcaacttctc ttttccaaac attataatgg aaaccccttg
  114661	cttttttttt tttaatttgc atttgcatga gagtttattt agctcggcat tttattttta
  114721	aaatttgtgt atatattttt gctatatatc tgtaacttat aaacagcaaa ttattggatt
  114781	ttgctttctg attctttctg taattcttct tacataagaa gttctcctat gagtaacatt
  114841	gctgtttaga gtgaggcatg atttatttcc agcttagtat gtattgggtc ggttaacccc
  114901	caaaggtcat gctcatcccc gccccatctc tgtgagttat tgtccgagtg tggagcgccc
  114961	tgtctaggcc gacgagagac ccaccatcgg gcacacctgc ccctcctggt ctggtcagtg
  115021	ccgggctctg tcctgagtcc actcctgatg tcacaggctg gtgcttcagc gacctcggct
  115081	gtgacacgga gggtgtgatg gcactgccca gccccatggg gcttggagga ctaaaggatg
  115141	cacacctgcc tggcagactg agggcacagg tgtttctcac actgtcagcg ttttgaaata
  115201	ttcctttgat tttctaccct aactcccaaa ggccgttcaa cataagctag aatgctacgt
  115261	ggtgcttgat tacattttag aaaagtttca gcaaatacca cgagatgcag caaagaacta
  115321	gacctcacag atcaggccgc ctgcataagg gagcccacac agtcgtggga gacggggacc
  115381	ctctcccacg tcctgtctgt cccaggatgg tcccctcacc cgccccctct ctcccctcgc
  115441	cctcctgtgg tgggggccgg ccaccatcac agctgcagag cctcaagaag ggggtcgccc
  115501	tggccactcc cgtggcagga gggacacgag ggcaggagct taccgcgggt gcagtggtct
  115561	cggatcagct cagctggccg ctgcggggtc ggggggacag ttcagtggga ggcaggagcc
  115621	cccactacag ctgccaggac ttctcagagg tgacaagggg gttcagtcac ctcagcccag
  115681	gtggaaacca aatggcctct tgcgcggctc ctggggccac gcggaggttc gctgggatca
  115741	caggtatctg gatgtgtgcg ccatggacat gcaccacctt cggggggtaa ggggtgggga
  115801	aaggcagccc ctttcttttg ggggaccccc tcttcagtgt ctgataacca ggaaaccaaa
  115861	tcagaaggtg gtctgggggt gctgagcagg gtgtctccta caccacaggc cacacactca
  115921	cacagcctcc aggactccag tggggctgag cgctggagac tcacccacgt ttgctacccc
  115981	cccacccaag gccatcccag aacagctgcc tgcgtcctca cggctggccc ctcccctctg
  116041	gtctaaccca gtgtgggtgg gccggcctgg ggtctccacc tgcctcctgc tgttccctgg
  116101	gctgctggct gtctgcagat gcggggccct ggcccggaga agccccatca gagcccagag
  116161	gacgggagtg gagcggggag gtgagccccg gagtctcgag gggccagagg caaaatactg
  116221	ggctgtgtcc ctggaaggca gtttcccatg aaaccttcaa tataggccgc cccagacgat
  116281	cagcctcatc tgctacgtgg attcctcccc gtagcgaatg gtgattgggt tctacatgga
  116341	cccgggactt ctgtttgaat tataatcttt cccccactgc ccctccaggg atctggaaaa
  116401	tggaggcctg ggctagacgg aagcttcctc caagattctt tattgaaggg attcgaagag
  116461	aaacaggtgg tcagtaatct gtgggggatg gaggggtgag cgctacgtgt aacggtttta
  116521	ctgttgctac gggaccagtt ttgatgtctt tccccttcaa gaagcagacc caaacaccga
  116581	gatgctgagg ttagcagcac agagcgggtt catccacaag gcaaccaggc agggagacca
  116641	gagacgctct ggaatctgcc tccctatggg cacgggctgg gtgctcacgg atgaagacca
  116701	agcagcaggt ggcgtggggc gtggggagcc tgcggaaagc gatggacaag gtgcgggacc
  116761	gcggtccgcg cggtggaccc aagctccgcc tctgcgctgc agcgcgagct gggggcggag
  116821	cttccaggga cccgcgaccg cgcccagtgg gagggtccgc ggtccaccca gtcctaacag
  116881	ctcagctcca gctagacgcc gctgagtccg gctttctaga gagcaacccc ggcgggtatt
  116941	ttatggttct ggcttcctga ttggaggaca cgcgagtctt agaacaccct tgattagtgc
  117001	gggcaggcgg aatggatttg actgatcacg atctgcagtt tcaccatctc aggggccgcc
  117061	ctcaccccca cctatcctgc caaagggggg gcctcggtgc tgagatcggg gccacacgtg
  117121	cactagacgg tcggtcagcg ctgctgctga gcggacccgg ggccatcctc acaccgccac
  117181	tggcccctgt gctcaataaa aggaaggaaa gcgggaaaag cgctttctgg ccgcggtggc
  117241	ctcgcgcgtt cctccatcgc catctgctgg cagagcccgg catggcaccc gctgcacaga
  117301	aacctcggtg tccgtttggg tgccccatcc ttgaccccga gagagcaccc tccgtccaaa
  117361	atgaaaaaca gctgctccca agagtcatta taatcacagc caattgtgtt aattcgtcct
  117421	cggatccact cacagttcca cggaacattc tgctaacctc tgacaactcc tacataaagc
  117481	aatactgaga agaaaagaac gtggttgata aatacaaagg catacaacaa taaggagcaa
  117541	agaaaaaaga cagtcctcgc agttctgttt tgttcatctc tcatgagtag gatggcagat
  117601	aaaacacaga atgcccagtg aataatttta gtctaagtat gtccccaata ctgcctaatc
  117661	ttcaaatcta accttatttt taaaatatat attttttgct ggtcactcat cagttcatgc
  117721	accaaagcct ttgtttcttg actcctaact ttttgacccc tctggggtga ggagcacccc
  117781	taacctcgag agcccatcac acagtcccct tgggactaga cccttctttg cccatcacag
  117841	ctgaccggaa gggccagccc atggccagcg ctcgcgcccc ctggcggaca gactctgcgc
  117901	ggcagccccg ggagcccagg tgcgaccccg cggtctctgg cgccctctag tgtggaaaga
  117961	tctcctcctg gtgttcccag tcattgggct gtattttatt agagaagatg ctcgcgtgac
  118021	gatgatgatg gtcctttacc gggaggcacg tttggggcgc gtcggctcag gggccgagct
  118081	attagcctgc atcgcgccca caggcatcgc gtccccctga gccgggtcag ctgtgggctg
  118141	tcctgacacg ggtttccccc agtctctggc ccgctgtccc tcccaggtca gtgtccagcg
  118201	ttgcccttct ggttgtggac ttgtgcagcg gtctcagcag atggaggggc gaccctaaag
  118261	gatgtattga ggcatctcag cactgtcctc cgcccaggtt tgctggtcag cagtgaagtg
  118321	accgggaaaa ggggctgtct tggggtcctt tcagaggcct gggttagacc aaagttttct
  118381	agaagattca ccattgcagg gagtcaaaga caaaactagg gtggtcagca atctgtgggg
  118441	gattcggcgg tgagggaatt ctgaatgcta catgtaatgg ttttactatt gttagggaac
  118501	atttttcccc cctacaaaca gcaggccaaa atactgagat gtcaggtttg catcaaagag
  118561	cgggttcatc cacaaggcaa ccagagaacg ctctggaatc tgcctccctg cgggcacagg
  118621	ctgggtgctc acggatgaag accaagcagc aggtggcgtg gggagtgggg agcctgggga
  118681	aagcgatgga caaggtgcga ggacctccgg cgcgagctgg aggcggagct tccagggaca
  118741	cgcggccacg cccagtggga gggtcagcgg tccatccagt cctaacagct cagctccaac
  118801	tagacgctgc tgagtctggc tttctagaga acactccggg cgggtatttt attgttttgg
  118861	cttcgtgact ggaggacgtt caagtcttaa aacacccttg attagtgcgg ggaggcggaa
  118921	tggatttgac tgatcacgac ccgcagtttc accatctcag gggccgccct caccccctcc
  118981	taccctacca aaggtggggg catcggtgct gagatctggg gtgacacata aaatcaggtg
  119041	aagtcttagg acagggggcc gattccaggt cctagggtgc agaaaaaacc tacctggccc
  119101	cgggctagac agcgtggagg gcgtggcccg ggctggtgca cagaagtggc ccccaactgg
  119161	tcagaaggtg tgggagccca gggctggtct actgcagaag gggtcgcctg gtggacagag
  119221	tggggcctga gtgcctgctg aactggtccg tcagggctgc tgagcagaca cgggccatca
  119281	tcactggctc ctgtgctcga tagaagggag ggaaaccagg aaagcaaagg cgctttatgg
  119341	ccgcttttgt gtttcgcgtt cctctagcac cgtctgccgg cagaacgcgg cattacatcc
  119401	gctggccaaa cctcggggtc cggcttggat gtccccatcc ttgtctcgga gatctcacct
  119461	ctcagcagtt cccctgggga caatgtcgag aagatgcgac cttgacccgg agctcggtgg
  119521	agagggtgcc ctgggttctt tccgcagttg cttggagtgg aggtgcctca tgttgggctg
  119581	ggaacgggag gaaggaaaca ggtcatgatt gagatgctct agacagactg tccctgctct
  119641	tgccaaattt cagaagattg tctttaataa atattccatt ttttgtatgc ccttaggtct
  119701	atttccagac actttaaata tattgaaaga ctttaaatat ttatataaaa atattattta
  119761	tagactgtat aaaaggaaca gttagaactg gacttggaac aacagactgg ttccaaatag
  119821	gaaaaggagt acgtcaaggc tgtatattgt caccctgctt atttaactta tatgcagagt
  119881	acatcatgag aaacgctggg ctggaagaaa cacaagctgg aatcaagatt gccgggagaa
  119941	atatcaataa cctcagatat gcagatgaca ccacccttat ggcagaaagt gaagaggaac
  120001	tcaaaagcct cttgatgaag gtgaaagagg agagcgaaaa agttggctta aagctcaaca
  120061	tttagaaaac gaagatcatg gcatctggtc ccatcacttc atggaaatag atggggaaac
  120121	agttgagaca gtgtcagact ttatttttgg gggctccaat gaaattaaaa gacgcttact
  120181	tcttggaagg aaagttatga ccaacctaga cagcatatta aaaagcagag acactacttt
  120241	gccagcaaag gtccgtctag tcaaggctat ggtttttcca gtggtcatgt atggatgtga
  120301	gagttggact gtgaagaagg ctgagcaccg aagaagtgat gcttttgaac tgtggtgttg
  120361	gagaagactc ttgagaggcc cttggactgc aaggagatcc aaccagtcca tcgtaaagga
  120421	gatcaccccc tgggtggtca ttggaaggac tgatgttgaa gctgaaactc cagtactttg
  120481	gctacctaat gcgaagagct gactcattgg aaaagaccct gatgctggga aagattgaag
  120541	gtgggaggag aaggggacaa cagaggatga gatggttgga ttgcatcact gactcgatgg
  120601	acgtgagtct gagtgaagtc tgggagttgg tgatggccag ggaggccctg gcgtgctggc
  120661	ggttcatggg gtcgcaaaga gtcggccatg actgagtgac tgaactgaac tgatccagaa
  120721	atttaaaatt aatatataaa ccaaatccat gcagacaatt ataagcatat attataaatg
  120781	cataattata agcaagtata tgttatattt ataatagttt ataatgtatt tataagcaag
  120841	tatatattat tataagcata attgtaagta gaagtaactt tgggctttcc tggtggctca
  120901	gacagtaaag aatctgcctg cagtacagga gaccgggttc gatccctggt ttggggaaat
  120961	tccctggaga agggaatggc aaccaactcc aacatgtttg cctggagaat tccatggaca
  121021	gaggagcccg gaaggttgca gtccatgggg ttgcaaagag ctggatacaa cagagtgact
  121081	aacacatgta tataaataaa tttacctata tattgtatat atatttataa acatattcag
  121141	atattataaa taattagaaa catattatac atgtatttaa atactgttat aaacataaat
  121201	ttaaaaaata attttcagcc ctttggcttg ggggtgtgtt tgtggacgtc tttgtgctac
  121261	tgttcctgaa gtggagctct cccctcccaa accagctttt gaaatgactg ggaaagcaat
  121321	ggaatacata agcatcagga agatagcaac agagctgtca ttcttcacag agggtgtgct
  121381	tgagtgtgta gcaagtcccg cagaatgtag acagattaat atagtctatt aaaaatagtg
  121441	tagcaaattt acgaggtgcg atttcaagta taaagactta ctgggtctct cagttcagtt
  121501	cagtcgcttg gttgtgtccg actctttttg accccatgga ccgcagcacg ccaggcctcc
  121561	ctgtccatca ccaactcctg gagttcactc aaactcatgt ccatcgagtc ggtgatgcca
  121621	tccaaccatc tcatcctctg gcgtcccctt ctcctcccac cttcaatctt tcccagcatc
  121681	agggtctttc ccagtgagtc agttctttgc atcaggtggc cagagtagtg gagtttcagc
  121741	ttcagcatcg gtccttccaa tgaatattct ggactgattt cctttaggat tgactggttg
  121801	gatctccttg cagttcaagg gactctcaag agtcttctcc aacagcacag tctatgaata
  121861	gaatagcaaa tgaatagaga ataacattta cgaggatata ttttaccatt gcataaaata
  121921	tatcagcttg tagagaacag acttgttccc aggggagagg gtgggtaggg atggagtggg
  121981	agtttgngat cancagaagc gagctgttat atagaagatg gataaaaagg atacacaaca
  122041	atgtcctact gtgtggcacc gggacctata ttcagtagct tgtgagaaac cataatcgac
  122101	aagactgagg aaaagtatat atatatgtat gtacttgagt tgctttgctg tacagaagaa
  122161	attaacacaa cattgtaaat cgatatttca atagaatcca cccccccaaa tatataagtt
  122221	tcctggagat ggagacggca acccactcca tttcttgcac ccaatattct tgcctggagg
  122281	atcccatgga tagaggatcg caaagactcg gacataaccc agcgactaac actttccctt
  122341	tcaaatgtgt aggtttacta gcgtgaatct acagagatgc ccaagacatt cgtttatgag
  122401	gaaaactcca cacgcagctt cactgagaat tattaaacct attaaaggga gagagcgcca
  122461	ggatattcat ggattgaaag attcgatgtg gtcaagttgc cagttttccc caaactgatt
  122521	ggtaaattcc ccaggagctg gctcaaggcg caaaattccc tttacctttt tttaagagac
  122581	gaagccaagg agccgattct ggttgagaga cgctcaggtc ctcctgcggg agagcagccc
  122641	tcttcctccc ggtcgcctgg gcagtttcga ggccacgacc agaaggactt ggctccctgt
  122701	gtcgcgcact cagaagtctc cctctccgtc ccaaggactc agaagctggg cgtcctgccc
  122761	gcagcagagg aggcagcctg gaggggcccc gcgggcacag cggtccgggt ttcagccgag
  122821	ttgcccgccc cgcccctcta cctgggcgct gccgcccggc tccggggccg gccgtgccct
  122881	ccgtggccgc aaggcgtcgc tgtccccccg ctggaagtgc tgacccggag gaaggggccc
  122941	agacggaggg actcggagcc tccgagtgac accctgggac tccgagcgct ggagcctggc
  123001	gtcaccccag gcaggggcag tgggggcccg gggcggggtc aggggcctcc cccggttctc
  123061	atttgacacc gcgggggtgc gctgggcaca gtgtccaggg gccacgttcc gagcaggggc
  123121	gcgatgcagg cccgggcgcg gcctgtcccg ggcgcgagtc cagctgcttt gcagaggtgg
  123181	cggcaggtcg cagtgaccct cacagagacg ccccactctg cggctccagg tgggcctgtg
  123241	ccccccagaa gtgctgacct gtgcaccggg aaggcacagg gccccccagc catgtctgcg
  123301	atggaagagc cggaaccgcg ccatgcccgt cctcgctgac cggcaggcac ccgccgtgtg
  123361	tccacacgct gagccatctg gctccccttg cttgacatac acccaggacc tgagtgtgca
  123421	ggaagttaga aggggcaggt gtggtgacac gatgccatcc agcatcacct gagaacctgg
  123481	acaaacctca ggggcccagc ctgctctgtg aggccccgag ggccggcccc tccccggacc
  123541	cctgccttga atccggccac actgcccgcc ttcctgctcc tgcggcttgt cagacacgcc
  123601	tgagcccagg gcctgtgcac tcgctgtccc ttctgccagg actgctcctc cccaggctct
  123661	tgctggggct ccccttcttc attcgggggt ggcctctctt gttcagtggc tcagctgtgc
  123721	ccagtctttg caaccccatg gactgcagca cgccaggctt ccctgtcctt cactagctcc
  123781	tggagtttgc tcaaactcat gtccattgag tcagtgatgc tatccaacca tctcatcctt
  123841	tgctgcccac ttcttctcct gctctcaatc tttcccagca tcagggtctt ttccaatgag
  123901	ttagctctct gcatcaggag gccaaagtat tggagcttca gcatcagtcc ttccagtgaa
  123961	tatgcgaggt tgatttccct tagaattgac tggttggatc tccttcctgt ccagagaact
  124021	ctcaagagtc ttctccagca ccacagtcgg agagcatcag ttcttcagtg atcaggtttc
  124081	tttatagccc agctctcaca tcggtacatg actattggaa aacccatagc tttgattaga
  124141	tggaccttca ttggcaaagt gatgggcctt cattggccct gctttttaat acaccatcta
  124201	ggtttgtcgt agctttcctt ccaaagagca aacatctttt aatttcctgg ctgcagtaac
  124261	catccatagt gattttggag cccaagaaaa taaaatctgc cactgtttcc actttttccc
  124321	cttctatttg ctatgaagtg aggggactgg atgccatgat cttagtttaa accagcagtt
  124381	gtcaccccga ccgcttcctt tcctaaagag ctcatcacac ctcccactgg aatgcaatgt
  124441	gttgcctgtc cgcctgcttc acctcctggg actttgctgc aggtcttggt ctctgaggcc
  124501	cctgccgtat ccccagggcc cagagcagtg ctgggcttcg agtccgatca gggactatgt
  124561	gtgtggactg gatggtgctt gcttcttctg gggaacgaga gacctgggcc tggggaacga
  124621	ggggacctgg tgtgaccgga tctcctccct cgggagagga gccaagcgag tggacacagg
  124681	tcagtgtgtc ttgctcctgt gtggcaggtg tcccgtctgt gtctgtcatc ttggcatttc
  124741	ggtgtttctg tgaacccagc ccctcccctc ctgatacccc atcccatcag cacagaggag
  124801	actgggcttg gggactctct ggtcctgaga ttcctctccg catgtgactc ccccctcctg
  124861	gggggagcag gcaccgtgtg tgaggagggt ggaagctttt caagaccccc agcttttctg
  124921	tcccaggggg ctctggcagg gccttgggag ctggaatgag ctggaatctg ggccagtggg
  124981	ggtttccctg gtggtaaaga acccgcctgc ccatgcacga ggcataagag acgcgggttc
  125041	gatcactggg tcgggaagat cccctacagg agggcatggc aacccactcc agtattcttt
  125101	cctgaagaat cccttggaca gaggagcctg gtgggctaca gtctctgggg tggcaaggag
  125161	tcggacacga ctgaagcgac ttaccatgca cgcacgcggg gtcaggggtc agggccgcgc
  125221	tgcttacctg ctgtgtgacc ttagccaggt cacacccccc aggctgtgaa agagaacagt
  125281	cttcccagac tcgggcatcc aggtctttac agacgtgcct gtgagctttg tgactctggc
  125341	tctgtggccg ctagagggcg ctgtccgccg ggccctatgt gcgtgcacgc atgtgagcat
  125401	gttcgcatac gtgtgtgcat ctgtcggggg cgcacggtgc ggggacacgg gcacgcggtc
  125461	aggaacgcag cccggacacc tccacgtggc ccgcgagtac cgtcaggtgg gggctgtggc
  125521	tccgctgtgt gggtgacccg ccctcccccc gcgaacgtgg tgcatagtga ccgcctggct
  125581	gggctcctga gctcagccat cctgcccccc gggtcagctc ccgacaggcc cagctctagg
  125641	ccccaggcgt ggaccgaggc ccccaggccc cggcctgtga gatgggacct ccgtctgggg
  125701	ggctcattct gctcccggag gcctggcagg cccctcctct ttggcattgc ataccctcgc
  125761	attggggtgg gtaagcacag taccccatgc ctgtggcccc gtgggagcgg cctgctcagg
  125821	gaggccggag cctcagctac agggctgtca caccgggctg cagaggaaga agacgggagc
  125881	gaggcctaca ggaacctagc caggccctgg cccactgagc cgacaggagc ctggccagag
  125941	gcctgcacag gacggggtgg cggggggggt ggggtggggt gctgggcccc gtggccttga
  126001	ctgcagaccc cgagggctcc tcagcttaga acggccaagc ctgagtcttg ggggtgcagg
  126061	tcaggggg

• Primers
• In another embodiment, primers are provided to generate 3′ and 5′ sequences of a targeting vector. The oligonucleotide primers can be capable of hybridizing to porcine immunoglobulin genomic sequence, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. In a particular embodiment, the primers hybridize under stringent conditions to Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. Another embodiment provides oligonucleotide probes capable of hybridizing to porcine heavy chain, kappa light chain or lambda light chain nucleic acid sequences, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The polynucleotide primers or probes can have at least 14 bases, 20 bases, 30 bases, or 50 bases which hybridize to a polynucleotide of the present invention. The probe or primer can be at least 14 nucleotides in length, and in a particular embodiment, are at least 15, 20, 25, 28, or 30 nucleotides in length.
• In one embodiment, primers are provided to amplify a fragment of porcine Ig heavy-chain that includes the functional joining region (the J6 region). In one non-limiting embodiment, the amplified fragment of heavy chain can be represented by Seq ID No 4 and the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 2, to produce the 5′ recombination arm and complementary to a portion of Ig heavy-chain mu constant region, such as, but not limited to Seq ID No 3, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 4) can be subcloned and assembled into a targeting vector.
• In other embodiments, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the constant region. In another embodiment, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the J region. In one non-limiting embodiment, the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 21 or 10, to produce the 5′ recombination arm and complementary to genomic sequence 3′ of the constant region, such as, but not limited to Seq ID No 14, 24 or 18, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 20) can be subcloned and assembled into a targeting vector.
II. Genetic Targeting of the Immunoglobulin Genes
• The present invention provides cells that have been genetically modified to inactivate immunoglobulin genes, for example, immunoglobulin genes described above. Animal cells that can be genetically modified can be obtained from a variety of different organs and tissues such as, but not limited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and a disaggregated preparation of a whole or part of an embryo, fetus, or adult animal. In one embodiment of the invention, cells can be selected from the group consisting of, but not limited to, epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, Islets of Langerhans cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, egg cells, leydig cells, peritubular cells, sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, squamous epithelial cells, osteocytes, osteoblasts, and osteoclasts. In one alternative embodiment, embryonic stem cells can be used. An embryonic stem cell line can be employed or embryonic stem cells can be obtained freshly from a host, such as a porcine animal. The cells can be grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF).
• In a particular embodiment, the cells can be fibroblasts; in one specific embodiment, the cells can be fetal fibroblasts. Fibroblast cells are a suitable somatic cell type because they can be obtained from developing fetuses and adult animals in large quantities. These cells can be easily propagated in vitro with a rapid doubling time and can be clonally propagated for use in gene targeting procedures.
• Targeting Constructs
• Homologous Recombination
• In one embodiment, immunoglobulin genes can be genetically targeted in cells through homologous recombination. Homologous recombination permits site-specific modifications in endogenous genes and thus novel alterations can be engineered into the genome. In homologous recombination, the incoming DNA interacts with and integrates into a site in the genome that contains a substantially homologous DNA sequence. In non-homologous (“random” or “illicit”) integration, the incoming DNA is not found at a homologous sequence in the genome but integrates elsewhere, at one of a large number of potential locations. In general, studies with higher eukaryotic cells have revealed that the frequency of homologous recombination is far less than the frequency of random integration. The ratio of these frequencies has direct implications for “gene targeting” which depends on integration via homologous recombination (i.e. recombination between the exogenous “targeting DNA” and the corresponding “target DNA” in the genome).
• A number of papers describe the use of homologous recombination in mammalian cells. Illustrative of these papers are Kucherlapati et al., Proc. Natl. Acad. Sci. USA 81:3153-3157, 1984; Kucherlapati et al., Mol. Cell. Bio. 5:714-720, 1985; Smithies et al, Nature 317:230-234, 1985; Wake et al., Mol. Cell. Bio. 8:2080-2089, 1985; Ayares et al., Genetics 111:375-388, 1985; Ayares et al., Mol. Cell. Bio. 7:1656-1662, 1986; Song et al., Proc. Natl. Acad. Sci. USA 84:6820-6824, 1987; Thomas et al. Cell 44:419-428, 1986; Thomas and Capecchi, Cell 51: 503-512, 1987; Nandi et al., Proc. Natl. Acad. Sci. USA 85:3845-3849, 1988; and Mansour et al., Nature 336:348-352, 1988. Evans and Kaufman, Nature 294:146-154, 1981; Doetschman et al., Nature 330:576-578, 1987; Thoma and Capecchi, Cell 51:503-512, 4987; Thompson et al., Cell 56:316-321, 1989.
• The present invention can use homologous recombination to inactivate an immunoglobulin gene in cells, such as the cells described above. The DNA can comprise at least a portion of the gene(s) at the particular locus with introduction of an alteration into at least one, optionally both copies, of the native gene(s), so as to prevent expression of functional immunoglobulin. The alteration can be an insertion, deletion, replacement or combination thereof. When the alteration is introduce into only one copy of the gene being inactivated, the cells having a single unmutated copy of the target gene are amplified and can be subjected to a second targeting step, where the alteration can be the same or different from the first alteration, usually different, and where a deletion, or replacement is involved, can be overlapping at least a portion of the alteration originally introduced. In this second targeting step, a targeting vector with the same arms of homology, but containing a different mammalian selectable markers can be used. The resulting transformants are screened for the absence of a functional target antigen and the DNA of the cell can be further screened to ensure the absence of a wild-type target gene. Alternatively, homozygosity as to a phenotype can be achieved by breeding hosts heterozygous for the mutation.
• Targeting Vectors
• In another embodiment, nucleic acid targeting vector constructs are also provided. The targeting vectors can be designed to accomplish homologous recombination in cells. These targeting vectors can be transformed into mammalian cells to target the ungulate heavy chain, kappa light chain or lambda light chain genes via homologous recombination. In one embodiment, the targeting vectors can contain a 3′ recombination arm and a 5′ recombination arm (i.e. flanking sequence) that is homologous to the genomic sequence of ungulate heavy chain, kappa light chain or lambda light chain genomic sequence, for example, sequence represented by Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The homologous DNA sequence can include at least 15 bp, 20 bp, 25 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence, particularly contiguous sequence, homologous to the genomic sequence. The 3′ and 5′ recombination arms can be designed such that they flank the 3′ and 5′ ends of at least one functional variable, joining, diversity, and/or constant region of the genomic sequence. The targeting of a functional region can render it inactive, which results in the inability of the cell to produce functional immunoglobulin molecules. In another embodiment, the homologous DNA sequence can include one or more intron and/or exon sequences. In addition to the nucleic acid sequences, the expression vector can contain selectable marker sequences, such as, for example, enhanced Green Fluorescent Protein (eGFP) gene sequences, initiation and/or enhancer sequences, poly A-tail sequences, and/or nucleic acid sequences that provide for the expression of the construct in prokaryotic and/or eukaryotic host cells. The selectable marker can be located between the 5′ and 3′ recombination arm sequence.
• Modification of a targeted locus of a cell can be produced by introducing DNA into the cells, where the DNA has homology to the target locus and includes a marker gene, allowing for selection of cells comprising the integrated construct. The homologous DNA in the target vector will recombine with the chromosomal DNA at the target locus. The marker gene can be flanked on both sides by homologous DNA sequences, a 3′ recombination arm and a 5′ recombination arm. Methods for the construction of targeting vectors have been described in the art, see, for example, Dai et al., Nature Biotechnology 20: 251-255, 2002; WO 00/51424.
• Various constructs can be prepared for homologous recombination at a target locus. The construct can include at least 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence homologous with the target locus. The sequence can include any contiguous sequence of an immunoglobulin gene.
• Various considerations can be involved in determining the extent of homology of target DNA sequences, such as, for example, the size of the target locus, availability of sequences, relative efficiency of double cross-over events at the target locus and the similarity of the target sequence with other sequences.
• The targeting DNA can include a sequence in which DNA substantially isogenic flanks the desired sequence modifications with a corresponding target sequence in the genome to be modified. The substantially isogenic sequence can be at least about 95%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to the corresponding target sequence (except for the desired sequence modifications). In a particular embodiment, the targeting DNA and the target DNA can share stretches of DNA at least about 75, 150 or 500 base pairs that are 100% identical. Accordingly, targeting DNA can be derived from cells closely related to the cell line being targeted; or the targeting DNA can be derived from cells of the same cell line or animal as the cells being targeted.
• Porcine Heavy Chain Targeting
• In particular embodiments of the present invention, targeting vectors are provided to target the porcine heavy chain locus. In one particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the J6 region of the porcine immunoglobulin heavy chain locus. Since the J6 region is the only functional joining region of the porcine immunoglobulin heavy chain locus, this will prevent the expression of a functional porcine heavy chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the J6 region, optionally including J1-4 and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the J6 region, including the mu constant region (a “J6 targeting construct”), see for example, FIG. 1. Further, this J6 targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No 5 and FIG. 1. In other particular embodiments, the 5′ targeting arm can contain sequence 5′ of J1, such as depicted in Seq ID No. 1 and/or Seq ID No 4. In another embodiments, the 5′ targeting arm can contain sequence 5′ of J1, J2 and/or J3, for example, as depicted in approximately residues 1-300, 1-500, 1-750, 1-1000 and/or 1-1500 Seq ID No 4. In a further embodiment, the 5′ targeting arm can contain sequence 5′ of the constant region, for example, as depicted in approximately residues 1-300, 1-500, 1-750, 1-1000, 1-1500 and/or 1-2000 or any fragment thereof of Seq ID No 4 and/or any contiguous sequence of Seq ID No. 4 or fragment thereof. In another embodiment, the 3′ targeting arm can contain sequence 3′ of the constant region and/or including the constant region, for example, such as resides 7000-8000 and/or 8000-9000 or fragment thereof of Seq ID No 4. In other embodiments, targeting vector can contain any contiguous sequence or fragment thereof of Seq ID No 4. sequence In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the diversity region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the diversity region of the porcine heavy chain locus. In a further embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the mu constant region and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the mu constant region of the porcine heavy chain locus.
• In further embodiments, the targeting vector can include, but is not limited to any of the following sequences: the Diversity region of heavy chain is represented, for example, by residues 1089-1099 of Seq ID No 29 (D(pseudo)), the Joining region of heavy chain is represented, for example, by residues 1887-3352 of Seq ID No 29 (for example: J(psuedo): 1887-1931 of Seq ID No 29, J(pseudo): 2364-2411 of Seq ID No 29, J(pseudo): 2756-2804 of Seq ID No 29, J (functional J): 3296-3352 of Seq ID No 29), the recombination signals are represented, for example, by residues 3001-3261 of Seq ID No 29 (Nonamer), 3292-3298 of Seq ID No 29 (Heptamer), the Constant Region is represented by the following residues: 3353-9070 of Seq ID No 29 (J to C mu intron), 5522-8700 of Seq ID No 29 (Switch region), 9071-9388 of Seq ID No 29 (Mu Exon 1), 9389-9469 of Seq ID No 29 (Mu Intron A), 9470-9802 of Seq ID No 29 (Mu Exon 2), 9830-10069 of Seq ID No 29 (Mu Intron B), 10070-10387 of Seq ID No 29 (Mu Exon 3), 10388-10517 of Seq ID No 29 (Mu Intron C), 10815-11052 of Seq ID No 29 (Mu Exon 4), 11034-11039 of Seq ID No 29 (Poly(A) signal) or any fragment or combination thereof. Still further, any contiguous sequence at least about 17, 20, 30, 40, 50, 100, 150, 200 or 300 nucleotides of Seq ID No 29 or fragment and/or combination thereof can be used as targeting sequence for the heavy chain targeting vector. It is understood that in general when designing a targeting construct one targeting arm will be 5′ of the other targeting arm.
• In other embodiments, targeting vectors designed to disrupt the expression of porcine heavy chain genes can contain recombination arms, for example, the 3′ or 5′ recombination arm, that target the constant region of heavy chain. In one embodiment, the recombination arm can target the mu constant region, for example, the C mu sequences described above or as disclosed in Sun & Butler Immunogenetics (1997) 46: 452-460. In another embodiment, the recombination arm can target the delta constant region, such as the sequence disclosed in Zhao et al. (2003) J imunol 171: 1312-1318, or the alpha constant region, such as the sequence disclosed in Brown & Butler (1994) Molec Immunol 31: 633-642.

• Seq ID No. 5

  GGCCAGACTTCCTCGGAACAGCTCAAAGAGCTCTGTCAAAGCCAGATCCC

  ATCACACGTGGGCACCAATAGGCCATGCCAGCCTCCAAGGGCCGAACTGG

  GTTCTCCACGGCGCACATGAAGCCTGCAGCCTGGCTTATCCTCTTCCGTG

  GTGAAGAGGCAGGCCCGGGACTGGACGAGGGGCTAGCAGGGTGTGGTAGG

  CACCTTGCGCCCCCCACCCCGGCAGGAACCAGAGACCCTGGGGCTGAGAG

  TGAGCCTCCAAACAGGATGCCCCACCCTTCAGGCCACCTTTCAATCCAGC

  TACACTCCACCTGCCATTCTCCTCTGGGCACAGGGCCCAGCCCCTGGATC

  TTGGCCTTGGCTCGACTTGCACCCACGCGCACACACACACTTCCTAACGT

  GCTGTCCGCTCACCCCTCCCCAGCGTGGTCCATGGGCAGCACGGCAGTGC

  GCGTCCGGCGGTAGTGAGTGCAGAGGTCCCTTCCCCTCCCCCAGGAGCCC

  CAGGGGTGTGTGCAGATCTGGGGGCTCCTGTCCCTTACACCTTCATGCCC

  CTCCCCTCATACCCACCCTCCAGGCGGGAGGCAGCGAGACCTTTGCCCAG

  GGACTCAGCCAACGGGCACACGGGAGGCCAGCCCTCAGCAGCTGGCTCCC

  AAAGAGGAGGTGGGAGGTAGGTCCACAGCTGCCACAGAGAGAAACCCTGA

  CGGACCCCACAGGGGCCACGCCAGCCGGAACCAGCTCCCTCGTGGGTGAG

  CAATGGCCAGGGCCCCGCCGGCCACCACGGCTGGCCTTGCGCCAGCTGAG

  AACTCACGTCCAGTGCAGGGAGACTCAAGACAGCCTGTGCACACAGCCTC

  GGATCTGCTCCCATTTCAAGCAGAAAAAGGAAACCGTGCAGGCAGCCCTC

  AGCATTTCAAGGATTGTAGCAGCGGCCAACTATTCGTCGGCAGTGGCCGA

  TTAGAATGACCGTGGAGAAGGGCGGAAGGGTCTCTCGTGGGCTCTGCGGC

  CAACAGGCCCTGGCTCCACCTGCCCGCTGCCAGCCCGAGGGGCTTGGGCC

  GAGCCAGGAACCACAGTGCTCACCGGGACCACAGTGACTGACCAAACTCC

  CGGCCAGAGCAGCCCCAGGCCAGCCGGGCTCTCGCCCTGGAGGACTCACC

  ATCAGATGCACAAGGGGGCGAGTGTGGAAGAGACGTGTCGCCCGGGCCAT

  TTGGGAAGGCGAAGGGACCTTCCAGGTGGACAGGAGGTGGGACGCACTCC

  AGGCAAGGGACTGGGTCCCCAAGGCCTGGGGAAGGGGTACTGGCTTGGGG

  GTTAGCCTGGCCAGGGAACGGGGAGCGGGGCGGGGGGCTGAGCAGGGAGG

  ACCTGACCTCGTGGGAGCGAGGCAAGTCAGGCTTCAGGCAGCAGCCGCAC

  ATCCCAGACCAGGAGGCTGAGGCAGGAGGGGCTTGCAGCGGGGCGGGGGC

  CTGCCTGGCTCCGGGGGCTCCTGGGGGACGCTGGCTCTTGTTTCCGTGTC

  CCGCAGCACAGGGCCAGCTCGCTGGGCCTATGCTTACCTTGATGTCTGGG

  GCCGGGGCGTCAGGGTCGTCGTCTCCTCAGGGGAGAGTCCCCTGAGGCTA

  CGCTGGGG*GGGGACTATGGCAGCTCCACCAGGGGCCTGGGGACCAGGGG

  CCTGGACCAGGCTGCAGCCCGGAGGACGGGCAGGGCTCTGGCTCTCCAGC

  ATCTGGCCCTCGGAAATGGCAGAACCCCTGGCGGGTGAGCGAGCTGAGAG

  CGGGTCAGACAGACAGGGGCCGGCCGGAAAGGAGAAGTTGGGGGCAGAGC

  CCGCCAGGGGCCAGGCCCAAGGTTCTGTGTGCCAGGGCCTGGGTGGGCAC

  ATTGGTGTGGCCATGGCTACTTAGACGCGTGATCAAGGGCGAATTCCAGC

  ACACTGGCGGCCGTTACTAGTggatcccggcgcgccctaccgggtagggg

  aggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctg

  ggcacttggcgctacacaagtggcctctggcctcgcacacattccacatc

  caccggtaggcgccaaccggctccgttctttggtggccccttcgcgccac

  cttctactcctcccctagtcaggaagttcccccccgccccgcagctcgcg

  tcgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgca

  gatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcg

  gccaatagcagctttggctccttcgctttctgggctcagaggctgggaag

  gggtgggtccgggggcgggctcaggggcgggctcaggggcggggcgggcg

  cccgaaggtcctccggaagcccggcattctgcacgcttcaaaagcgcacg

  tctgccgcgctgttctcctcttcctcatctccgggcctttcgacctgcag

  ccaatatgggatcggccattgaacaagatggattgcacgcaggttctccg

  gccgcttgggtggagaggctattcggctatgactgggcacaacagacaat

  cggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccgg

  ttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggac

  gaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagc

  tgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcg

  aagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaa

  gtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggc

  tacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgta

  ctcggatggaagccggtcttgtcaatcaggatgatctggacgaagagcat

  caggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcc

  cgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaata

  tcatggtggaaaatggccgcttttctggattcatcgactgtggccggctg

  ggtgtggcggatcgctatcaggacatagcgttggctacccgtgatattgc

  tgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggta

  tcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgag

  ttcttctgaggggatcaattcTCTAGATGCATGCTCGAGCGGCCGCCAGT

  GTGATGGATATCTGCAGAATTCGCCCTtCCAGGCGTTGAAGTCGTCGTGT

  CCTCAGGTAAGAACGGCCCTCCAGGGCCTTTAATTTCTGCTCTCGTCTGT

  GGGCTTTTCTGACTCTGATCCTCGGGAGGCGTCTGTGCCCCCCCCGGGGA

  TGAGGCCGGCTTGCCAGGAGGGGTCAGGGACCAGGAGCCTGTGGGAAGTT

  CTGACGGGGGCTGCAGGCGGGAAGGGCCCCACCGGGGGGCGAGCCCCAGG

  CCGCTGGGCGGCAGGAGACCCGTGAGAGTGCGCCTTGAGGAGGGTGTCTG

  CGGAACCACGAACGCCCGCCGGGAAGGGCTTGCTGCAATGCGGTCTTCAG

  ACGGGAGGCGTCTTCTGCCCTCACCGTCTTTCAAGCCCTTGTGGGTCTGA

  AAGAGCCATGTCGGAGAGAGAAGGGACAGGCCTGTCCCGACCTGGCCGAG

  AGCGGGCAGCCCCGGGGGAGAGCGGGGCGATCGGCCTGGGCTCTGTGAGG

  CCAGGTCCAAGGGAGGACGTGTGGTCCTCGTGACAGGTGCACTTGCGAAA

  CCTTAGAAGACGGGGTATGTTGGAAGCGGCTCCTGATGTTTAAGAAAAGG

  GAGACTGTAAAGTGAGCAGAGTCCTCAAGTGTGTTAAGGTTTTAAAGGTC

  AAAGTGTTTTAAACCTTTGTGACTGCAGTTAGCAAGCGTGCGGGGAGTGA

  ATGGGGTGCCAGGGTGGCCGAGAGGCAGTACGAGGGCCGTGCCGTCCTCT

  AATTCAGGGCTTAGTTTTGCAGAATAAAGTCGGCCTGTTTTCTAAAAGCA

  TTGGTGGTGCTGAGCTGGTGGAGGAGGCCGCGGGCAGCCCTGGCCACCTG

  CAGCAGGTGGCAGGAAGCAGGTCGGCCAAGAGGCTATTTTAGGAAGCCAG

  AAAACACGGTCGATGAATTTATAGCTTCTGGTTTCCAGGAGGTGGTTGGG

  CATGGCTTTGCGCAGCGCCACAGAACCGAAAGTGCCCACTGAGAAAAAAC

  AACTCCTGCTTAATTTGCATTTTTCTAAAAGAAGAAACAGAGGCTGACGG

  AAACTGGAAAGTTCCTGTTTTAACTACTCGAATTGAGTTTTCGGTCTTAG

  CTTATCAACTGCTCACTTAGATTCATTTTCAAAGTAAACGTTTAAGAGCC

  GAGGCATTCCTATCCTCTTCTAAGGCGTTATTCCTGGAGGCTCATTCACC

  GCCAGCACCTCCGCTGCCTGCAGGCATTGCTGTCACCGTCACCGTGACGG

  CGCGCACGATTTTCAGTTGGCCCGCTTCCCCTCGTGATTAGGACAGACGC

  GGGCACTCTGGCCCAGCCGTCTTGGCTCAGTATCTGCAGGCGTCCGTCTC

  GGGACGGAGCTCAGGGGAAGAGCGTGACTCCAGTTGAACGTGATAGTCGG

  TGCGTTGAGAGGAGACCCAGTCGGGTGTCGAGTCAGAAGGGGCCCGGGGC

  CCGAGGCCCTGGGCAGGACGGCCCGTGCCCTGCATCACGGGCCCAGCGTC

  CTAGAGGCAGGACTCTGGTGGAGAGTGTGAGGGTGCCTGGGGCCCCTCCG

  GAGCTGGGGCCGTGCGGTGCAGGTTGGGCTCTCGGCGCGGTGTTGGCTGT

  TTCTGCGGGATTTGGAGGAATTCTTCCAGTGATGGGAGTCGCCAGTGACC

  GGGCACCAGGCTGGTAAGAGGGAGGCCGCCGTCGTGGCCAGAGCAGCTGG

  GAGGGTTCGGTAAAAGGCTCGCCCGTTTCCTTTAATGAGGACTTTTCCTG

  GAGGGCATTTAGTCTAGTCGGGACCGTTTTCGACTCGGGAAGAGGGATGC

  GGAGGAGGGCATGTGCCCAGGAGCCGAAGGCGCCGCGGGGAGAAGCCCAG

  GGCTCTCCTGTCCCCACAGAGGCGACGCCACTGCCGCAGACAGACAGGGC

  CTTTCCCTCTGATGACGGCAAAGGCGCCTCGGCTCTTGCGGGGTGCTGGG

  GGGGAGTCGCCCCGAAGCCGCTCACCCAGAGGCCTGAGGGGTGAGACTGA

  CCGATGCCTCTTGGCCGGGCCTGGGGCCGGACCGAGGGGGACTCCGTGGA

  GGCAGGGCGATGGTGGCTGCGGGAGGGAACCGACCCTGGGCCGAGCCCGG

  CTTGGCGATTCCCGGGCGAGGGCCCTCAGCCGAGGCGAGTGGGTCCGGCG

  GAACCACCCTTTCTGGCCAGCGCCACAGGGCTCTCGGGACTGTCCGGGGC

  GACGCTGGGCTGCCCGTGGCAGGCCTGGGCTGACCTGGACTTCACCAGAC

  AGAACAGGGCTTTCAGGGCTGAGCTGAGCCAGGTTTAGCGAGGCCAAGTG

  GGGCTGAACCAGGCTCAACTGGCCTGAGCTGGGTTGAGCTGGGCTGACCT

  GGGCTGAGCTGAGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCT

  GGACTGGCTGAGCTGAGCTGGGTTGAGCTGAGCTGAGCTGGCCTGGGTTG

  AGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGTTGAGCTGGGTTG

  ATCTGAGCTGAGCTGGGCTGAGCTGAGCTAGGCTGGGGTGAGCTGGGCTG

  AGCTGGTTTGAGTTGGGTTGAGCTGAGCTGAGCTGGGCTGTGCTGGCTGA

  GCTAGGCTGAGCTAGGCTAGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAG

  GCTGGGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCGTTGAGCTGG

  CTGGGCTGGATTGAGCTGGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCT

  GGGTTGAGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGGGTTGAGCT

  GTCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCT

  CAGCAGAGCTGGGTTGGGCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCT

  GGCCTGGGTTGAGCTGGGCTGAGCTGAGCTGGGCTGAGCTGGCCTGTGTT

  GAGCTGGGCTGGGTTGAGCTGGGCTGAGCTGGATTGAGCTGGGTTGAGCT

  GAGCTGGGCTGGGCTGTGCTGACTGAGCTGGGCTGAGCTAGGCTGGGGTG

  AGCTGGGCTGAGCTGATCCGAGCTAGGCTGGGCTGGTTTGGGCTGAGCTG

  AGCTGAGCTAGGCTGGATTGATCTGGCTGAGCTGGGTTGAGCTGAGCTGG

  GCTGAGCTGGTCTGAGCTGGCCTGGGTCGAGCTGAGCTGGACTGGTTTGA

  GCTGGGTCGATCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGA

  GCTGAGCTGGGTTGAGCTGGGCTGAGCTGAGGGCTGGGGTGAGCTGGGCT

  GAACTAGCCTAGCTAGGTTGGGCTGAGCTGGGCTGGTTTGGGCTGAGCTG

  AGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCAGGCCTGG

  GGTGAGCTGGGCTAGGTGGAGCTGAGCTGGGTCGAGCTGAGTTGGGCTGA

  GCTGGCCTGGGTTGAGGTAGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGA

  GCTGGCTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGG

  TTGAGCTGGGCTCGGTTGAGCTGGGCTGAGCTGAGCCGACCTAGGCTGGG

  ATGAGCTGGGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAG

  CAGGCTGAGCTGGGCCTGGAGCCTGGCCTGGGGTGAGCTGGGCTGAGCTG

  CGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAA

  GCTGGGCCGAGCTGGCCTGGGATGAGCTGGGCCGGTTTGGGCTGAGCTGA

  GCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCTGGCCTGGG

  GTGAGCTGGGCTGAGCTAAGCTGAGCTGGGCTGGTTTGGGCTGAGCTGGC

  TGAGCTGGGTCCTGCTGAGCTGGGCTGAGCTGACCAGGGGTGAGCTGGGC

  TGAGTTAGGCTGGGCTCAGCTAGGCTGGGTTGATCTGGCAGGGCTGGTTT

  GCGCTGGGTCAAGCTCCCGGGAGATGGCCTGGGATGAGCTGGGCTGGTTT

  GGGCTGAGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTG

  GGCTGAGCTGGCCTGGGGTGAGCTGGGCTGGGTGGAGCTGAGCTGGGCTG

  AACTGGGCTAAGCTGGCTGAGCTGGATCGAGCTGAGCTGGGCTGAGCTGG

  CCTGGGGTTAGCTGGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTGG

  CTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGTTGAG

  CTGGGCTGGGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGGCTGGGCTGAG

  CTGAGCTAGGCTGCATTGAGCTGGCTGGGATGGATTGAGCTGGCTGAGCT

  GGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGG

  GTTGAGCTGAGCTGGGCTGAGCTGGGCTCAGCAGAGCTGGGTTGAGCTGA

  GCTGGGTTGAGCTGGGGTGAGCTGGGCTGAGCAGAGCTGGGTTGAGCTGA

  GCTGGGTTGAGCTGGGCTCGAGCAGAGCTGGGTTGAGCTGAGCTGGGTTG

  AGCTGGGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTG

  AGCTAGCTGGGCTCAGCTAGGCTGGGTTGAGCTGAGCTGGGCTGAACTGG

  GCTGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCTGGGCTGAGCAGA

  GCTGGGCTGAGCAGAGCTGGGTTGGTCTGAGCTGGGTTGAGCTGGGCTGA

  GCTGGGCTGAGCAGAGTTGGGTTGAGCTGAGCTGGGTTCAGCTGGGCTGA

  GCTAGGCTGGGTTGAGCTGGGTTGAGTTGGGCTGAGCTGGGCTGGGTTGA

  GCGGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCGGAACTGGGTTGA

  TCTGAATTGAGCTGGGCTGAGCCGGGCTGAGCCGGGCTGAGCTGGGCTAG

  GTTGAGCTTGGGTGAGCTTGCCTCAGCTGGTCTGAGCTAGGTTGGGTGGA

  GCTAGGCTGGATTGAGCTGGGCTGAGCTGAGCTGATCTGGCCTCAGCTGG

  GCTGAGGTAGGCTGAACTGGGCTGTGCTGGGCTGAGCTGAGCTGAGCCAG

  TTTGAGCTGGGTTGAGCTGGGCTGAGCTGGGCTGTGTTGATCTTTCCTGA

  ACTGGGCTGAGCTGGGCTGAGCTGGCCTAGCTGGATTGAACGGGGGTAAG

  CTGGGCCAGGCTGGACTGGGCTGAGCTGAGCTAGGCTGAGCTGAGTTGAA

  TTGGGTTAAGCTGGGCTGAGATGGGCTGAGCTGGGCTGAGCTGGGTTGAG

  CCAGGTCGGACTGGGTTACCCTGGGCCACACTGGGCTGAGCTGGGCGGAG

  CTCGATTAACCTGGTCAGGCTGAGTCGGGTCCAGCAGACATGCGCTGGCC

  AGGCTGGCTTGACCTGGACACGTTCGATGAGCTGCCTTGGGATGGTTCAC

  CTCAGCTGAGCCAGGTGGCTCCAGCTGGGCTGAGCTGGTGACCCTGGGTG

  ACCTCGGTGACCAGGTTGTCCTGAGTCCGGGCCAAGCCGAGGCTGCATCA

  GACTCGCCAGACCCAAGGCCTGGGCCCCGGCTGGCAAGCCAGGGGCGGTG

  AAGGCTGGGCTGGCAGGACTGTCCCGGAAGGAGGTGCACGTGGAGCCGCC

  CGGACCCCGACCGGCAGGACCTGGAAAGACGCCTCTCACTCCCCTTTCTC

  TTCTGTCCCCTCTCGGGTCCTCAGAGAGCCAGTCTGCCCCGAATCTCTAC

  CCCCTCGTCTCCTGCGTCAGCCCCCCGTCCGATGAGAGCCTGGTGGCCCT

  GGGCTGCCTGGCCCGGGACTTCCTGCCCAGCTCCGTCACCTTCTCCTGGA

  A

• Porcine Kappa Chain Targeting
• In particular embodiments of the present invention, targeting vectors are provided to target the porcine kappa chain locus. In one particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the constant region of the porcine immunoglobulin kappa chain locus. Since the present invention discovered that there is only one constant region of the porcine immunoglobulin kappa light chain locus, this will prevent the expression of a functional porcine kappa light chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the constant region, optionally including the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the constant region, optionally including at least part of the enhancer region (a “Kappa constant targeting construct”), see for example, FIG. 2. Further, this kappa constant targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No 20 and FIG. 2. In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the joining region of the porcine kappa light chain locus. In other embodiments, the 5′ arm of the targeting vector can include Seq ID No 12 and/or Seq ID No 25 or any contiguous sequence or fragment thereof. In another embodiment, the 3′ arm of the targeting vector can include Seq ID No 15, 16 and/or 19 or any contiguous sequence or fragment thereof.
• In further embodiments, the targeting vector can include, but is not limited to any of the following sequences: the coding region of kappa light chain is represented, for example by residues 1-549 of Seq ID No 30 and 10026-10549 of Seq ID No 30, whereas the intronic sequence is represented, for example, by residues 550-10025 of Seq ID No 30, the Joining region of kappa light chain is represented, for example, by residues 5822-7207 of Seq ID No 30 (for example, J1:5822-5859 of Seq ID No 30, J2:6180-6218 of Seq ID No 30, J3:6486-6523 of Seq ID No 30, J4:6826-6863 of Seq ID No 30, J5:7170-7207 of Seq ID No 30), the Constant Region is represented by the following residues: 10026-10549 of Seq ID No 30 (C exon) and 10026-10354 of Seq ID No 30 (C coding), 10524-10529 of Seq ID No 30 (Poly(A) signal) and 11160-11264 of Seq ID No 30 (SINE element) or any fragment or combination thereof. Still further, any contiguous sequence at least about 17, 20, 30, 40, 50, 100, 150, 200 or 300 nucleotides of Seq ID No 30 or fragment and/or combination thereof can be used as targeting sequence for the heavy chain targeting vector. It is understood that in general when designing a targeting construct one targeting arm will be 5′ of the other targeting arm.

• Seq ID No. 20

  ctcaaacgtaagtggctttttccgactgattctttgctgtttctaattgt

  tggttggctttttgtccatttttcagtgttttcatcgaattagttgtcag

  ggaccaaacaaattgccttcccagattaggtaccagggaggggacattgc

  tgcatgggagaccagagggtggctaatttttaacgtttccaagccaaaat

  aactggggaagggggcttgctgtcctgtgagggtaggtttttatagaagt

  ggaagttaaggggaaatcgctatggttcacttttggctcggggaccaaag

  tggagcccaaaattgagtacattttccatcaattatttgtgagatttttg

  tcctgttgtgtcatttgtgcaagtttttgacattttggttgaatgagcca

  ttcccagggacccaaaaggatgagaccgaaaagtagaaaagagccaactt

  ttaagctgagcagacagaccgaattgttgagtttgtgaggagagtagggt

  ttgtagggagaaaggggaacagatcgctggctttttctctgaattagcct

  ttctcatgggactggcttcagagggggtttttgatgagggaagtgttcta

  gagccttaactgtgggttgtgttcggtagcgggaccaagctggaaatcaa

  acgtaagtgcacttttctactcctttttctttcttatacgggtgtgaaat

  tggggacttttcatgtttggagtatgagttgaggtcagttctgaagagag

  tgggactcatccaaaaatctgaggagtaagggtcagaacagagttgtctc

  atggaagaacaaagacctagttagttgatgaggcagctaaatgagtcagt

  tgacttgggatccaaatggccagacttcgtctgtaaccaacaatctaatg

  agatgtagcagcaaaaagagatttccattgaggggaaagtaaaattgtta

  atattgtggatcacctttggtgaagggacatccgtggagattgaacgtaa

  gtattttttctctactaccttctgaaatttgtctaaatgccagtgttgac

  ttttagaggcttaagtgtcagttttgtgaaaaatgggtaaacaagagcat

  ttcatatttattatcagtttcaaaagttaaactcagctccaaaaatgaat

  ttgtagacaaaaagattaatttaagccaaattgaatgattcaaaggaaaa

  aaaaattagtgtagatgaaaaaggaattcttacagctccaaagagcaaaa

  gcgaattaattttctttgaactttgccaaatcttgtaaatgatttttgtt

  ctttacaatttaaaaaggttagagaaatgtatttcttagtctgttttctc

  tcttctgtctgataaattattatatgagataaaaatgaaaattaatagga

  tgtgctaaaaaatcagtaagaagttagaaaaatatatgtttatgttaaag

  ttgccacttaattgagaatcagaagcaatgttatttttaaagtctaaaat

  gagagataaactgtcaatacttaaattctgcagagattctatatcttgac

  agatatctcctttttcaaaaatccaatttctatggtagactaaatttgaa

  atgatcttcctcataatggagggaaaagatggactgaccccaaaagctca

  gattt*aagaaaacctgtttaag*gaaagaaaataaaagaactgcatttt

  ttaaaggcccatgaatttgtagaaaaataggaaatattttaataagtgta

  ttcttttattttcctgttattacttgatggtgtttttataccgccaagga

  ggccgtggcaccgtcagtgtgatctgtagaccccatggcggccttttttc

  gcgattgaatgaccttggcggtgggtccccagggctctggtggcagcgca

  ccagccgctaaaagccgctaaaaactgccgctaaaggccacagcaacccc

  gcgaccgcccgttcaactgtgctgacacagtgatacagataatgtcgcta

  acagaggagaatagaaatatgacgggcacacgctaatgtggggaaaagag

  ggagaagcctgatttttattttttagagattctagagataaaattcccag

  tattatatccttttaataaaaaatttctattaggagattataaagaattt

  aaagctatttttttaagtggggtgtaattctttcagtagtctcttgtcaa

  atggatttaagtaatagaggcttaatccaaatgagagaaatagacgcata

  accctttcaaggcaaaagctacaagagcaaaaattgaacacagcagccag

  ccatctagccactcagattttgatcagttttactgagtttgaagtaaata

  tcatgaaggtataattgctgataaaaaaataagatacaggtgtgacacat

  ctttaagtttcagaaatttaatggcttcagtaggattatatttcacgtat

  acaaagtatctaagcagataaaaatgccattaatggaaacttaatagaaa

  tatatttttaaattccttcattctgtgacagaaattttctaatctgggtc

  ttttaatcacctaccctttgaaagagtttagtaatttgctatttgccatc

  gctgtttactccagctaatttcaaaagtgatacttgagaaagattatttt

  tggtttgcaaccacctggcaggactattttagggccattttaaaactctt

  ttcaaactaagtattttaaactgttctaaaccatttagggccttttaaaa

  atcttttcatgaatttcaaacttcgttaaaagttattaaggtgtctggca

  agaacttccttatcaaatatgctaatagtttaatctgttaatgcaggata

  taaaattaaagtgatcaaggcttgacccaaacaggagtatcttcatagca

  tatttcccctcctttttttctagaattcatatgattttgctgccaaggct

  attttatataatctctggaaaaaaaatagtaatgaaggttaaaagagaag

  aaaatatcagaacattaagaattcggtattttactaactgcttggttaac

  atgaaggtttttattttattaaggtttctatctttataaaaatctgttcc

  cttttctgctgatttctccaagcaaaagattcttgatttgttttttaact

  cttactctcccacccaagggcctgaatgcccacaaaggggacttccagga

  ggccatctggcagctgctcaccgtcagaagtgaagccagccagttcctcc

  tgggcaggtggccaaaattacagttgacccctcctggtctggctgaacct

  tgccccatatggtgacagccatctggccagggcccaggtctccctctgaa

  gcctttgggaggagagggagagtggctggcccgatcacagatgcggaagg

  ggctgactcctcaaccggggtgcagactctgcagggtgggtctgggccca

  acacacccaaagcacgcccaggaaggaaaggcagcttggtatcactgccc

  agagctaggagaggcaccgggaaaatgatctgtccaagacccgttcttgc

  ttctaaactccgagggggtcagatgaagtggttttgtttcttggcctgaa

  gcatcgtgttccctgcaagaagcggggaacacagaggaaggagagaaaag

  atgaactgaacaaagcatgcaaggcaaaaaaggGGGTCTAGCCGCGGTCT

  AGGAAGCTTTCTAGGGTACCTCTAGGGATCCCGGCGCGCCCTACCGGGTA

  GGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCC

  GCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCA

  CATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCG

  CCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCT

  CGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCG

  TGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGC

  AGCGGCCAATAGCAGCTTTGGCTCCTTCGCTTTCTGGGCTCAGAGGCTGG

  GAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCG

  GGCGCCCGAAGGTCCTCCGGAAGCCCGGCATTCTGCACGCTTCAAAAGCG

  CACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCT

  GCAGCCAATATGGGATCGGCCATTGAACAAGATGGATTGCACGCAGGTTC

  TCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGA

  CAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGC

  CCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCA

  GGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCG

  CAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTG

  GGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGA

  GAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATC

  CGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCA

  CGTACTCGGATGGAAGCCGGTCTTGTCAATCAGGATGATCTGGACGAAGA

  GCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCA

  TGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCG

  AATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCG

  GCTGGGTGTGGCGGATCGCTATCAGGACATAGCGTTGGCTACCCGTGATA

  TTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTAC

  GGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGA

  CGAGTTCTTCTGAGGGGATCAATTCTCTAGAGCTCGCTGATCAGCCTCGA

  CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT

  TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA

  GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG

  GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT

  GCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTG

  GGGGCGCGCCCctcgagcggccgccagtgtgatggatatctgcagaattc

  gcccttggatcaaacacgcatcctcatggacaatatgttgggttcttagc

  ctgctgagacacaacaggaactcccctggcaccactttagaggccagaga

  aacagcacagataaaattccctgccctcatgaagcttatagtctagctgg

  ggagatatcataggcaagataaacacatacaaatacatcatcttaggtaa

  taatatatactaaggagaaaattacaggggagaaagaggacaggaattgc

  tagggtaggattataagttcagatagttcatcaggaacactgttgctgag

  aagataacatttaggtaaagaccgaagtagtaaggaaatggaccgtgtgc

  ctaagtgggtaagaccattctaggcagcaggaacagcgatgaaagcactg

  aggtgggtgttcactgcacagagttgttcactgcacagagttgtgtgggg

  aggggtaggtcttgcaggctcttatggtcacaggaagaattgttttactc

  ccaccgagatgaaggttggtggattttgagcagaagaataattctgcctg

  gtttatatataacaggatttccctgggtgctctgatgagaataatctgtc

  aggggtgggatagggagagatatggcaataggagccttggctaggagccc

  acgacaataattccaagtgagaggtggtgctgcattgaaagcaggactaa

  caagacctgctgacagtgtggatgtagaaaaagatagaggagacgaaggt

  gcatctagggttttctgcctgaggaattagaaagataaagctaaagctta

  tagaagatgcagcgctctggggagaaagaccagcagctcagttttgatcc

  atctggaattaattttggcataaagtatgaggtatgtgggttaacattat

  ttgttttttttttttccatgtagctatccaactgtcccagcatcatttat

  tttaaaagactttcctttcccctattggattgttttggcaccttcactga

  agatcaactgagcataaaattgggtctatttctaagctcttgattccatt

  ccatgacctatttgttcatctttaccccagtagacactgccttgatgatt

  aaagcccctgttaccatgtctgttttggacatggtaaatctgagatgcct

  attagccaaccaagcaagcacggcccttagagagctagatatgagagcct

  ggaattcagacgagaaaggtcagtcctagagacatacatgtagtgccatc

  accatgcggatggtgttaaaagccatcagactgcaacagactgtgagagg

  gtaccaagctagagagcatggatagagaaacccaagcactgagctgggag

  gtgctcctacattaagagattagtgagatgaaggactgagaagattgatc

  agagaagaaggaaaatcaggaaaatggtgctgtcctgaaaatccaaggga

  agagatgttccaaagaggagaaaactgatcagttgtcagctagcgtcaat

  tgggatgaaaatggaccattggacagagggatgtagtgggtcatgggtga

  atagataagagcagcttctatagaatggcaggggcaaaattctcatctga

  tcggcatgggttctaaagaaaacgggaagaaaaaattgagtgcatgacca

  gtcccttcaagtagagaggtggaaaagggaaggaggaaaatgaggccacg

  acaacatgagagaaatgacagcatttttaaaaattttttattttatttta

  tttatttatttttgctttttagggctgcccctgcaacatatggaggttcc

  caggttaggggtctaatcagagctatagctgccagcctacaccacagcca

  tagcaatgccagatctacatgacctacaccacagctcacagcaacgccgg

  atccttaacccactgagtgaggccagagatcaaacccatatccttatgga

  tactagtcaggttcattaccactgagccaaaatgggaaatcctgagtaat

  gacagcattttttaatgtgccaggaagcaaaacttgccaccccgaaatgt

  ctctcaggcatgtggattattttgagctgaaaacgattaaggcccaaaaa

  acacaagaagaaatgtggaccttcccccaacagcctaaaaaatttagatt

  gagggcctgttcccagaatagagctattgccagacttgtctacagaggct

  aagggctaggtgtggtggggaaaccctcagagatcagagggacgtttatg

  taccaagcattgacatttccatctccatgcgaatggccttcttcccctct

  gtagccccaaaccaccacccccaaaatcttcttctgtctttagctgaaga

  tggtgttgaaggtgatagtttcagccactttggcgagttcctcagttgtt

  ctgggtctttcctccTgatccacattattcgactgtgtttgattttctcc

  tgtttatctgtctcattggcacccatttcattcttagaccagcccaaaga

  acctagaagagtgaaggaaaatttcttccaccctgacaaatgctaaatga

  gaatcaccgcagtagaggaaaatgatctggtgctgcgggagatagaagag

  aaaatcgctggagagatgtcactgagtaggtgagatgggaaaggggtgac

  acaggtggaggtgttgccctcagctaggaagacagacagttcacagaaga

  gaagcgggtgtccgtggacatcttgcctcatggatgaggaaaccgaggct

  aagaaagactgcaaaagaaaggtaaggattgcagagaggtcgatccatga

  ctaaaatcacagtaaccaaccccaaaccaccatgttttctcctagtctgg

  cacgtggcaggtactgtgtaggttttcaatattattggtttgtaacagta

  cctattaggcctccatcccctcctctaatactaacaaaagtgtgagactg

  gtcagtgaaaaatggtcttctttctctatgaatctttctcaagaagatac

  ataactttttattttatcataggcttgaagagcaaatgagaaacagcctc

  caacctatgacaccgtaacaaaatgtttatgatcagtgaagggcaagaaa

  caaaacatacacagtaaagaccctccataatattgtgggtggcccaacac

  aggccaggttgtaaaagctttttattctttgatagaggaatggatagtaa

  tgtttcaacctggacagagatcatgttcactgaatccttccaaaaattca

  tgggtagtttgaattataaggaaaataagacttaggataaatactttgtc

  caagatcccagagttaatgccaaaatcagttttcagactccaggcagcct

  gatcaagagcctaaactttaaagacacagtcccttaataactactattca

  cagttgcactttcagggcgcaaagactcattgaatcctacaatagaatga

  gtttagatatcaaatctctcagtaatagatgaggagactaaatagcgggc

  atgacctggtcacttaaagacagaattgagattcaaggctagtgttcttt

  ctacctgttttgtttctacaagatgtagcaatgcgctaattacagacctc

  tcagggaaggaa

• Porcine Lambda Chain Targeting
• In particular embodiments of the present invention, targeting vectors are provided to target the porcine lambda chain locus. In one embodiment, lambda can be targeted by designing a targeting construct that contains a 5′ arm containing sequence located 5′ to the first JC unit and a 3′ arm containing sequence 3′ to the last JC unit of the J/C cluster region, thus preventing functional expression of the lambda locus (see, FIGS. 3-4). In one embodiment, the targeting vector can contain any contiguous sequence (such as about 17, 20, 30, 40, 50, 75, 100, 200, 300 or 5000 nucleotides of contiguous sequence) or fragment thereof. Seq ID No 28. In one embodiment, the 5′ targeting arm can contain Seq ID No. 32, which includes 5′ flanking sequence to the first lambda J/C region of the porcine lambda light chain genomic sequence or any contiguous sequence (such as about 17, 20, 30, 40, 50, 75, 100, 200, 300 or 5000 nucleotides of contiguous sequence) or fragment thereof (see also, for example FIG. 5). In another embodiment, the 3′ targeting arm can contain, but is not limited to one or more of the following: Seq ID No. 33, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, from approximately 200 base pairs downstream of lambda J/C; Seq ID No. 34, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, approximately 11.8 Kb downstream of the J/C cluster, near the enhancer; Seq ID No. 35, which includes approximately 12 Kb downstream of lambda, including the enhancer region; Seq ID No. 36, which includes approximately 17.6 Kb downstream of lambda; Seq ID No. 37, which includes approximately 19.1 Kb downstream of lambda; Seq ID No. 38, which includes approximately 21.3 Kb downstream of lambda; and Seq ID No. 39, which includes approximately 27 Kb downstream of lambda, or any contiguous sequence (such as about 17, 20, 30, 40, 50, 75, 100, 200, 300 or 5000 nucleotides of contiguous sequence) or fragment thereof of Seq ID Nos 32-39 (see also, for example FIG. 6). It is understood that in general when designing a targeting construct one targeting arm will be 5′ of the other targeting arm.
• Seq ID No. 48 (as shown in Example 4) provides a representative, non-limiting example of a targeting construct that contains a 5′ arm containing sequence located 5′ to the first JC unit and a 3′ arm containing sequence 3′ to the last JC unit of the J/C cluster region. Representative 5′ and 3′ arms are shown in Seq ID No. 49 and 50 (also in Example 4).
• In another embodiment, lambda is targeted using two targeting vectors. The two lambda targeting vectors, i.e., a vector pair, are utilized in a two step strategy to delete the entire J/C region of porcine lambda. In the first step, a first targeting vector is inserted upstream of the J/C region (or alternatively downstream of the J/C region). If the first targeting vector is inserted upstream of the J/C region, the 5′ and 3′ recombination arms of the first targeted vector contain homologous sequence to the 5′ flanking sequence of the first J/C unit of the J/C cluster region. See FIG. 5, which shows 7 JC units in the J/C cluster region. If the first targeting vector is inserted downstream of the J/C cluster region, the 5′ and 3′ recombination arms of the first targeting vector contain homologous sequence to the 3′ region of the last J/C unit in the JC region.
• The first-step vectors are designed with lox sites that flank a fusion gene which can provide both positive and negative selection. Selection of the targeting event utilizes the Tn5 APHII gene commonly described as Neo resistance. Once targeting events are isolated, Cre is provided transiently to facilitate deletion of the selectable marker located between two lox sites. Negative selection is then provided by the Herpes simplex thymidine kinase coding region. This step selects for targeted cells that have deleted the selectable marker and retains a single lox site upstream (alternatively downstream) of the J/C region.
• The second step is performed in the same lineage as the first step. The second targeting step also inserts a marker that provides both positive and negative selection. However, the second step inserts the marker on the opposite site of the J/C region in comparison to the first step. That is, if the first vector was inserted upstream of the J/C region, the second targeting vector is inserted downstream, and vice versa. FIG. 6 shows a second targeting vector inserted downstream of the J/C region. In addition, the second targeting vector has a single lox site that is located distally compared to the first vector. In other words, for the first strategy, the second vector has a single lox site located downstream of the marker gene (the alternative vector has the lox site upstream of the marker). After Cre mediated deletion, the region between the first targeting event (which left a lox remnant) and the second targeting event (which has a lox site outside of the marker) is deleted. Cells that have deleted the entire J/C cluster region are thus obtained.
• In a representative, non-limiting example, the vector pair is Seq. ID No. 44 (step 1) and Seq. ID No. 45 (step 2).
• In a further, non-limiting example, the vector pair is Seq. ID No. 46 (step 1) and Seq. ID No. 47 (step 2).

• SEQ. ID 44

  taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacg

  tcgctgagcaggccctggcctccctggccgagggcggtttgcgtattaga

  ggcctaaatggccgaattcagcggataacaatttcacacaggaaacagct

  atgaccatgattatctagtaactataacggtcctaaggtagcgagcgatc

  gcttaattaacctgcagggatatcccatgggggccgccagtgtgatggat

  atctgcagaattcgcccttgatattaagagaagggcaagtcagcttaagt

  ttgggggtagaggggaacagggagtgaggagatctggcctgagagatagg

  agccctggtggccacaggaggactctttgggtcctgtcggatggacacag

  ggcggcccgggggcatgttggagcccggctggttcttaccagaggcaggg

  ggcaccctctgacacgggagcagggcatgttccatacatgacacacccct

  ctgctccagggcaggtgggtggcggcacagaggagccagggactctgagc

  aaggggtccaccagtggggcagttggatccagacttctctgggccagcga

  gagtctagccctcagccgttctctgtccaggaggggggtggggcaggcct

  gggcggccagagctcatccctcaagggttcccagggtcctgccagaccca

  gatttccgaccgcagccaccacaagaggatgtggtctgctgtggcagctg

  ccaagaccttgcagcaggtgcagggtgggggggtgggggcacctgggggc

  agctggggtcactgagttcagggaaaaccccttttttcccctaaacctgg

  ggccatccctaggggaaaccacaacttctgagccctgggcagtggctgct

  gggagggaagagcttcatcctggaccctgggggggaacccagctccaaag

  gtgcaaggggcccaggtccaaggctagagtgggccaagcaccgcaatggc

  cagggagtgggggaggtggagctggactggatcagggcctccttgggact

  ccctacaccctgtgtgacatgttagggtacccacaccccatcaccagtca

  gggcctggcccatctccagggccagggatgtgcatgtaagtgtgtgtgag

  tgtgtgtgtgtggtgtagtacaccccttggcatccggttccgaggccttg

  ggttcctccaaagttgctctctgaattaggtcaaactgtgaggtcctgat

  cgccatcatcaacttcgttctccccacctcccatcattatcaagagctgg

  ggagggtctgggatttcttcccacccacaagccaaaagataagcctgctg

  gtgatggcagaagacacaggatcctgggtcagagacaaaggccagtgtgt

  cacagcgagagaggcagccggactatcagctgtcacagagaggccttagt

  ccgctgaactcaggccccagtgactcctgttccactgggcactggccccc

  ctccacagcgcccccaggccccagggagaggcgtcacagcttagagatgg

  ccctgctgaacagggaacaagaacaggtgtgccccatccagcgccccagg

  ggtgggacaggtgggctggatttggtgtgaagcccttgagccctggaacc

  caaccacagcagggcagttggtagatgccatttggggagaggccccagga

  gtaagggccatgggcccttgagggggccaggagctgaggacagggacaga

  gacggcccaggcagaggacagggccatgaggggtgcactgagatggccac

  tgccagcaggggcagctgccaacccgtccagggaacttattcagcagtca

  gctggaggtgccattgaccctgagggcagatgaagcccaggccaggctag

  gtgggctgtgaagaccccaggggacagagctctgtccctgggcagcactg

  gcctctcattctgcagggcttgacgggatcccaaggcctgctgcccctga

  tggtagtggcagtaccgcccagagcaggaccccagcatggaaaccccaac

  gggacgcagcctgcggagcccacaaaaccagtaaggagccgaagcagtca

  tggcacggggagtgtggacttccctttgatggggcccaggcatgaaggac

  agaatgggacagcggccatgagcagaaaatcagccggaggggatgggcct

  aggcagacgctggctttatttgaagtgttggcattttgtctggtgtgtat

  tgttggtattgattttattttagtatgtcagtgacatactgacatattat

  gtaacgacatattattatgtgttttaagaagcactccaagggaacaggct

  gtctgtaatgtgtccagagaagagagcaagagcttggctcagtctccccc

  aaggaggtcagttcctcaacaggggtcctaaatgtttcctggagccaggc

  ctgaatcaagggggtcatatctacacgtggggcagacccatggaccattt

  tcggagcaataagatggcagggaggataccaagctggtcttacagatcca

  gggctttgacctgtgacgcgggcgctcctccaggcaaagggagaagccag

  caggaagctttcagaactggggagaacagggtgcagacctccagggtctt

  gtacaacgcaccctttatcctggggtccaggaggggtcactgagggattt

  aagtgggggaccatcagaaccaggtttgtgttttggaaaaatggctccaa

  agcagagaccagtgtgaggccagattagatgatgaagaagaggcagtgga

  aagtcgatgggtggccaggtagcaagagggcctatggagttggcaagtga

  atttaaagtggtggcaccagagggcagatggggaggagcaggcactgtca

  tggactgtctatagaaatctaaaatgtataccctttttagcaatatgcag

  tgagtcataaaagaacacatatatatttcctttggccggccggcgcgcca

  cgcgtataacttcgtatagcatacattatacgaagttatcttaagggcta

  tggcagggcctgccgccccgacgttggctgcgagccctgggccttcaccc

  gaacttggggggtggggtggggaaaaggaagaaacgcgggcgtattggcc

  ccaatggggtctcggtggggtatcgacagagtgccagccctgggaccgaa

  ccccgcgtttatgaacaaacgacccaacaccgtgcgttttattctgtctt

  tttattgccgtcatagcgcgggttccttccggtattgtctccttccgtgt

  ttcactcgagttagaagaactcgtcaagaaggcgatagaaggcgatgcgc

  tgcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcagccca

  ttcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcct

  gatagcggtccgccacacccagccggccacagtcgatgaatccagaaaag

  cggccattttccaccatgatattcggcaagcaggcatcgccatgggtcac

  gacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagtt

  cggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgaca

  agaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttg

  gtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattg

  catcagccatgatggatactttctcggcaggagcaaggtgagatgacagg

  agatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttc

  agtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagcc

  acgatagccgcgctgcctcgtcctgcagttcattcagggcaccggacagg

  tcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacac

  ggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaata

  gcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttca

  atggccgatcccattccagatctgttagcctcccccatctcccgtgcaaa

  cgtgcgcgccaggtcgcagatcgtcggtatggagcctggggtggtgacgt

  gggtctggatcatcccggaggtaagttgcagcagggcgtcccggcagccg

  gcgggcgattggtcgtaatccaggataaagacgtgcatgggacggaggcg

  tttggtcaagacgtccaaggcccaggcaaacacgttgtacaggtcgccgt

  tgggggccagcaactcgggggcccgaaacagggtaaataacgtgtccccg

  atatggggtcgtgggcccgcgttgctctggggctcggcaccctggggcgg

  cacggccgtccccgaaagctgtccccaatcctcccgccacgacccgccgc

  cctgcagataccgcaccgtattggcaagcagcccgtaaacgcggcgaatc

  gcggccagcatagccaggtcaagccgctcgccggggcgctggcgtttggc

  caggcggtcgatgtgtctgtcctccggaagggcccccaacacgatgtttg

  tgccgggcaaggtcggcgggatgagggccacgaacgccagcacggcctgg

  ggggtcatgctgcccataaggtatcgcgcggccgggtagcacaggagggc

  ggcgatgggatggcggtcgaagatgagggtgagggccgggggcggggcat

  gtgagctcccagcctcccccccgatatgaggagccagaacggcgtcggtc

  acggcataaggcatgcccattgttatctgggcgcttgtcattaccaccgc

  cgcgtccccggccgatatctcaccctggtcaaggcggtgttgtgtggtgt

  agatgttcgcgattgtctcggaagcccccagcacccgccagtaagtcatc

  ggctcgggtacgtagacgatatcgtcgcgcgaacccagggccaccagcag

  ttgcgtggtggtggttttccccatcccgtggggaccgtctatataaaccc

  gcagtagcgtgggcattttctgctccgggcggacttccgtggcttcttgc

  tgccggcgagggcgcaacgccgtacgtcggttgctatggccgcgagaacg

  cgcagcctggtcgaacgcagacgcgtgctgatggccggggtacgaagcca

  tggtggctctagaggtcgaaaggcccggagatgaggaagaggagaacagc

  gcggcagacgtgcgcttttgaagcgtgcagaatgccgggcttccggagga

  ccttcgggcgcccgccccgcccctgagcccgcccctgagcccgcccccgg

  acccaccccttcccagcctctgagcccagaaagcgaaggagccaaagctg

  ctattggccgctgccccaaaggcctacccgcttccattgctcagcggtgc

  tgtccatctgcacgagactagtgagacgtgctacttccatttgtcacgtc

  ctgcacgacgcgagctgcggggcgggggggaacttcctgactaggggagg

  agtagaaggtggcgcgaaggggccaccaaagaacggagccggttggcgcc

  taccggtggatgtggaatgtgtgcgaggccagaggccacttgtgtagcgc

  caagtgcccagcggggctgctaaagcgcatgctccagactgccttgggaa

  aagcgcctcccctacccggtagggatccgcgttacataacttacggtaaa

  tggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataa

  tgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaa

  tgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgta

  tcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccg

  cctggcattatgcccagtacatgaccttatgggactttcctacttggcag

  tacatctacgtattagtcatcgctattaccatggtgatgcggttttggca

  gtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtc

  tccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgg

  ttaacaagcttataacttcgtatagcatacattatacgaagttattacgt

  agcggccgcgtcgacgataaattgtgtaattccacttctaaggattcatc

  ccaaggggggaaaataatcaaagatgtaaccaaaggtttacaaacaagaa

  ctcatcattaatcttccttgttgttatttcaacgatattattattattac

  tattattattattattattttgtctttttgcattttctagggccactccc

  acggcatagagaggttcccaggctaggggtcaaatcggagctacagctgc

  cggcctacgccagagccacagcaacgcaggatctgagccacagcaatgca

  ggatctacaccacagctcatggtaacgctggatccttaacccaatgagtg

  aggccagggatcgaacctgtaacttcatggttcctagtcggattcattaa

  ccactgagccacgacaggaactccaacattattaatgatgggagaaaact

  ggaagtaacctaaatatccagcagaaagggtgtggccaaatacagcatgg

  agtagccatcataaggaatcttacacaagcctccaaaattgtgtttctga

  aattgggtttaaagtacgtttgcattttaaaaagcctgccagaaaataca

  gaaaaatgtctgtgatatgtctctggctgataggattttgcttagtttta

  attttggctttataattttctatagttatgaaaatgttcacaagaagata

  tatttcattttagcttctaaaataattataacacagaagtaatttgtgct

  ttaaaaaaatattcaacacagaagtatataaagtaaaaattgaggagttc

  ccatcgtggctcagtgattaacaaacccaactagtatccatgaggatatg

  gatttgatccctggccttgctcagtgggttgaggatccagtgttgctgtg

  agctgtggtgtaggttgcagacacagcactctggcgttgctgtgactctg

  gcgtaggccggcagctacagctccatttggacccttagcctgggaacctc

  catatgcctgagatacggccctaaaaagtcaaaagccaaaaaaatagtaa

  aaattgagtgtttctacttaccacccctgcccacatcttatgctaaaacc

  cgttctccagagacaaacatcgtcaggtgggtctatatatttccagccct

  cctcctgtgtgtgtatgtccgtaaaacacacacacacacacacacacgca

  cacacacacacacgtatctaattagcattggtattagtttttcaaaaggg

  aggtcatgctctaccttttaggcggcaaatagattatttaaacaaatctg

  ttgacattttctatatcaacccataagatctcccatgttcttggaaaggc

  tttgtaagacatcaacatctgggtaaaccagcatggtttttagggggttg

  tgtggatttttttcatattttttagggcacacctgcagcatatggaggtt

  cccaggctaggggttgaatcagagctgtagctgccggcctacaccacagc

  cacagcaacgccagatccttaacccactgagaaaggccagggattgaacc

  tgcatcctcatggatgctggtcagatttatttctgctgagccacaacagg

  aactccctgaaccagaatgcttttaaccattccactttgcatggacattt

  agattgtttccatttaaaaatacaaattacaaggagttcccgtcgtggct

  cagtggtaacgaattggactaggaaccatgaggtttcgggttcgatccct

  ggccttgctcggtgggttaaggatccagcattgatgtgagatatggtgta

  ggtcgcagacgtggctcggatcccacgttgctgtggctctggcgtaggcc

  ggcaacaacagctccgattcgacccctagcctgggaacctccatgtgcca

  caggagcagccctagaaaaggcaaaaagacaaaaaaataaaaaattaaaa

  tgaaaaaataaaataaaaatacaaattacaagagacggctacaaggaaat

  ccccaagtgtgtgcaaatgccatatatgtataaaatgtactagtgtctcc

  tcgcgggaaagttgcctaaaagtgggttggctggacagagaggacaggct

  ttgacattctcataggtagtagcaatgggcttctcaaaatgctgttccag

  tttacactcaccatagcaaatgacagtgcctcttcctctccacccttgcc

  aataatgtgacaggtggatctttttctattttgtgtatctgacaagcaaa

  aaatgagaacaggagttcctgtcgtggtgcagtggagacaaatctgacta

  ggaaccatgaaatttcgggttcaatccctggcctcactcagtaggtaaag

  gatccagggttgcagtgagctgtggggtaggtcgcagacacagtgcaaat

  ttggccctgttgtggctgtggtgtaggccggcagctatagctccaattgg

  acccctagcctgggaacctccttatgccgtgggtgaggccctaaaaaaaa

  gagtgcaaaaaaaaaaaataagaacaaaaatgatcatcgtttaattcttt

  atttgatcattggtgaaacttattttccttttatatttttattgactgat

  tttatttctcctatgaatttaccggtcatagttttgcctgggtgttttta

  ctccggttttagttttggttggttgtattttcttagagagctatagaaac

  tcttcatctatttggaatagtaattcctcattaagtatttgtgctgcaaa

  aaattttccctgatctgttttatgcttttgtttgtggggtctttcacgag

  aaagcctttttagtttttacacctcagcttggttgtttttcttgattgtg

  tctgtaatctgcggccaacataggaaacacatttttactttagtgttttt

  ttcctattttcttcaagtacgtccattgttttggtgtctgattttacttt

  gcctggggtttgtttttgtgtggcaggaatataaacttatgtattttcca

  aatggagagccaatggttgtatatttgttgaattcaaatgcaactttatc

  aaacaccaaatcatcgatttatcacaactcttctctggtttattgatcta

  atgatcaattcctgttccacgctgttttaattattttagctttgtggatt

  ttggtgcctggtagagaacaaagcctccattattttcattcaaaatagtc

  ccgtctattatctgccattgttgtagtattagactttaaaatcaatttac

  tgattttcaaaagttattcctttggtgatgtggaatactttatacttcat

  aaggtacatggattcatttgtggggaattgatgtctttgctattgtggcc

  atttgtcaagttgtgtaatattttacccatgccaactttgcatattgtat

  gtgagtttattcccagggtttttaataggatgtttattgaagttgtcagt

  gtttccacaatttcatcgcctcagtgcttactgtttgcataaaaggaaac

  ctactcacttttgcctattgctcttgtattcaatcattttagttaactct

  tgtgttaattttgagagtttttcagctgactgtctggggttttctttaat

  agactagccctttgtctgtaaagaataattttatcgaatttttcttaaca

  ctcacactctccccacccccacccccgctcatctcctttcattgggtcaa

  atctgtagaatacaataaaagtaagagtgggaaccttagcctttaagtcg

  attttgcctttaaatgtgaatgttgctatgtttcgggacattctctttat

  caagttgcggatgtttccttagataattaacttaataaaagactggatgt

  ttgctttcttcaaatcagaattgtgttgaatttatattgctattctgttt

  aattttgtttcaaaaaatttacatgcacaccttaaagataaccatgacca

  aatagtcctcctgctgagagaaaatgttggccccaatgccacaggttacc

  tcccgactcagataaactacaatgggagataaaatcagatttggcaaagc

  ctgtggattcttgccataactctcagagcatgacttgggtgttttttcct

  tttctaagtattttaatggtatttttgtgttacaataggaaatctaggac

  acagagagtgattcaatgaggggaacgcattctgggatgactctaggcct

  ctggtttggggagagctctattgaagtaaagacaatgagaggaagcaagt

  ttgcagggaactgtgaggaatttagatggggaatgttgggtttgaggttt

  ctatagggcacgcaagcagagatgcactcaggaggaagaaggagcataaa

  tctagtggcgctgccggcaagcttgctggaggaggccaattgggagctgc

  tggaatgcatggaggcggcgctctcgaggctggaggaggccagctgattt

  aaatcggtccgcgtacgatgcatattaccctgttatccctaccgcggtta

  ctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgatgctct

  tctcccggtgaaaacctctgacacatggctcttctaaatccggagtttaa

  acgcttccttcatgtgagcaaaaggccagcaaaaggccaggaaccgtaaa

  aaggccgcgttgctggcgtttttccataggctccgcccccctgacgagca

  tcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactat

  aaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgtt

  ccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaag

  cgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtagg

  tcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgac

  cgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagaca

  cgacttatcgccactggcagcagccactggtaacaggattagcagagcga

  ggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggc

  tacactagaaggacagtatttggtatctgcgctctgctgaagccagttac

  cttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctg

  gtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaa

  ggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtg

  gaacgaaaactcacgttaagggattttggtcatgcctaggtggcaaacag

  ctattatgggtattatgggtctaccggtgcatgagattatcaaaaaggat

  cttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaa

  gtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgag

  gcacctatctcagcgatctgtctatttcgttcatccatagttgcctgact

  ccccgtcgtgtagataactacgatacgggagggcttaccatctggcccca

  gtgctgcaatgataccgcgagacccacgctcaccggctccagatttatca

  gcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaac

  tttatccgcctccatccagtctattaattgttgccgggaagctagagtaa

  gtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggc

  atcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttc

  ccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcgg

  ttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtg

  ttatcactcatggttatggcagcactgcataattctcttactgtcatgcc

  atccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattct

  gagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgg

  gataataccgcgccacatagcagaactttaaaagtgctcatcattggaaa

  acgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatcca

  gttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttact

  ttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaa

  aaagggaataagggcgacacggaaatgttgaatactcatactcttccttt

  ttcaatattattgaagcatttatcagggttattgtctcgggagcggatac

  atatttgaatgtatttagaaaaa

  SEQ ID 45

  taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacg

  tcgctgagcaggccctggcctccctggccgagggcggtttgcgtattaga

  ggcctaaatggccgaattcagcggataacaatttcacacaggaaacagct

  atgaccatgattatctagtaactataacggtcctaaggtagcgagcgatc

  gcttaattaacctgcagggataaccactgacccatgacgggaactcccag

  ggctcagctcttgactccaggttcgcagctgccctcaaagcaatgcaacc

  ctggctggccccgcctcatgcatccggcctcctccccaaagagctctgag

  cccacctgggcctaggtcctcctccctgggactcatggcctaagggtaca

  gagttactggggctgatgaagggaccaatggggacaggggcctcaaatca

  aagtggctgtctctctcatgtcccttcctctcctcagggtccaaaatcag

  ggtcagggccccagggcaggggctgagagggcctctttctgaaggccctg

  tctcagtgcaggttatgggggtctgggggagggtcaatgcagggctcacc

  cttcagtgccccaaagcctagagagtgagtgcctgccagtggcttcccag

  gcccaatcccttgactgcctgggaatgctcaaatgcaggaactgtcacaa

  caccttcagtcaggggctgctctgggaggaaaaacactcagaattggggg

  ttcagggaaggcccagtgccaagcatagcaggagctcaggtggctgcaga

  tggtgtgaaccccaggagcaggatggccggcactccccccagaccctcca

  gagccccaggttggctgccctcttcactgccgacacccctgggtccactt

  ctgccctttcccacctaaaacctttagggctcccactttctcccaaatgt

  gagacatcaccacggctcccagggagtgtccagaagggcatctggctgag

  aggtcctgacatctgggagcctcaggccccacaatggacagacgccctgc

  caggatgctgctgcagggctgttagctaggcggggtggagatggggtact

  ttgcctctcagaggccccggccccaccatgaaacctcagtgacaccccat

  ttccctgagttcacatacctgtatcctactccagtcaccttccccacgaa

  cccctgggagcccaggatgatgctggggctggagccacgaccagcccacg

  agtgatccagctctgccaatcagcagtcatttcccaagtgttccagccct

  gccaggtcccactacagcagtaatggaggccccagacaccagtccagcag

  ttagagggctggactagcaccagctttcaagcctcagcatctcaaggtga

  atggccagtgcccctccccgtggccatcacaggatcgcagatatgaccct

  aggggaagaaatatcctgggagtaaggaagtgcccatactcaaggatggc

  ccctctgtgacctaacctgtccctgaggattgtacttccaggcgttaaaa

  cagtagaacgcctgcctgtgaacccccgccaagggactgcttggggaggc

  cccctaaaccagaacacaggcactccagcaggacctctgaactctgacca

  ccctcagcaagtgggcaccccccgcagcttccaaggcaccccagggctca

  ccacagcggcccctcctggcagcccctcacccaggcccagaccctctaag

  atggcacatctaagccaatccacctccttgtcattcctcctgtccccacc

  caggacccttctcagatgaaaccttcgctccagccgctgggccctctctc

  ctgcccctctggcagttctccagggactccgcctcccactctctgtctct

  ccctgcactcctaggaacaagcgacctccaggaagcccagtccaattatc

  ccctctgtgtcctccccaatctctgcctctgggtggatttgagcaccaca

  tcctgttctcttcgacctgaaactccttggccccggtgtccgctctcctg

  ggccctcttttctctcctcccctcttccgtgccccgtttgtttggtgtta

  caggcaggccccggggagccgtccctccagctgctcttccttgtctgtct

  caggagccagaaactggcagcatctaaaaagggctcctgtttcttcatct

  gcccagcctcctagcccaaccagggctctggcctcactccagagggtggg

  ctccagagggcaggggttgcaccctcttagtgcctcagaggctcagctgg

  gtgcaggatgggggggccctcagggagcccctcagtgactgctgatcact

  tactgcaggactgttcccagctcttcccaatcattggaatgacaatacct

  agttctgctccatcatagtgatgcaggaaaaatgttactgaaatcctggt

  tcttgtttagcaatcgaagaatgaattccgcgaacacacaggcagcaagc

  aagcgaagcctttattaaaggaaagcagatagctcccagggctgcaggga

  gcggggagaagagctccccactctctattgtcctatagggctttttaccc

  cttaaagttggggggatacaaaaaaaatagaagaaaaagggagttcccgt

  cagggcacagcagaaacaaatccaactaggaaccatgaggttgggggttc

  gattcctggcctctctcagtgggttaaggatgcagcgttgccgtgagcta

  tgatacaggtcacagatgcagctcagatctactagtcaattgacaggcgc

  cggagcaggagctaggcctttggccggccggcgcgccagatctcttaagg

  gctatggcagggcctgccgccccgacgttggctgcgagccctgggccttc

  acccgaacttggggggtggggtggggaaaaggaagaaacgcgggcgtatt

  ggccccaatggggtctcggtggggtatcgacagagtgccagccctgggac

  cgaaccccgcgtttatgaacaaacgacccaacaccgtgcgttttattctg

  tctttttattgccgtcatagcgcgggttccttccggtattgtctccttcc

  gtgtttcactcgagttagaagaactcgtcaagaaggcgatagaaggcgat

  gcgctgcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcag

  cccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatg

  tcctgatagcggtccgccacacccagccggccacagtcgatgaatccaga

  aaagcggccattttccaccatgatattcggcaagcaggcatcgccatggg

  tcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaac

  agttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatc

  gacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcg

  cttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgc

  attgcatcagccatgatggatactttctcggcaggagcaaggtgagatga

  caggagatcctgccccggcacttcgcccaatagcagccagtcccttcccg

  cttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggcc

  agccacgatagccgcgctgcctcgtcctgcagttcattcagggcaccgga

  caggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccgga

  acacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccg

  aatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttg

  ttcaatggccgatcccattccagatctgttagcctcccccatctcccgtg

  caaacgtgcgcgccaggtcgcagatcgtcggtatggagcctggggtggtg

  acgtgggtctggatcatcccggaggtaagttgcagcagggcgtcccggca

  gccggcgggcgattggtcgtaatccaggataaagacgtgcatgggacgga

  ggcgtttggtcaagacgtccaaggcccaggcaaacacgttgtacaggtcg

  ccgttgggggccagcaactcgggggcccgaaacagggtaaataacgtgtc

  cccgatatggggtcgtgggcccgcgttgctctggggctcggcaccctggg

  gcggcacggccgtccccgaaagctgtccccaatcctcccgccacgacccg

  ccgccctgcagataccgcaccgtattggcaagcagcccgtaaacgcggcg

  aatcgcggccagcatagccaggtcaagccgctcgccggggcgctggcgtt

  tggccaggcggtcgatgtgtctgtcctccggaagggcccccaacacgatg

  tttgtgccgggcaaggtcggcgggatgagggccacgaacgccagcacggc

  ctggggggtcatgctgcccataaggtatcgcgcggccgggtagcacagga

  gggcggcgatgggatggcggtcgaagatgagggtgagggccgggggcggg

  gcatgtgagctcccagcctcccccccgatatgaggagccagaacggcgtc

  ggtcacggcataaggcatgcccattgttatctgggcgcttgtcattacca

  ccgccgcgtccccggccgatatctcaccctggtcaaggcggtgttgtgtg

  gtgtagatgttcgcgattgtctcggaagcccccagcacccgccagtaagt

  catcggctcgggtacgtagacgatatcgtcgcgcgaacccagggccacca

  gcagttgcgtggtggtggttttccccatcccgtggggaccgtctatataa

  acccgcagtagcgtgggcattttctgctccgggcggacttccgtggcttc

  ttgctgccggcgagggcgcaacgccgtacgtcggttgctatggccgcgag

  aacgcgcagcctggtcgaacgcagacgcgtgctgatggccggggtacgaa

  gccatggtggctctagaggtcgaaaggcccggagatgaggaagaggagaa

  cagcgcggcagacgtgcgcttttgaagcgtgcagaatgccgggcttccgg

  aggaccttcgggcgcccgccccgcccctgagcccgcccctgagcccgccc

  ccggacccaccccttcccagcctctgagcccagaaagcgaaggagccaaa

  gctgctattggccgctgccccaaaggcctacccgcttccattgctcagcg

  gtgctgtccatctgcacgagactagtgagacgtgctacttccatttgtca

  cgtcctgcacgacgcgagctgcggggcgggggggaacttcctgactaggg

  gaggagtagaaggtggcgcgaaggggccaccaaagaacggagccggttgg

  cgcctaccggtggatgtggaatgtgtgcgaggccagaggccacttgtgta

  gcgccaagtgcccagcggggctgctaaagcgcatgctccagactgccttg

  ggaaaagcgcctcccctacccggtagggatccgcgttacataacttacgg

  taaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtca

  ataatgacgtatgttcccatagtaacgccaatagggactttccattgacg

  tcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaag

  tgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatgg

  cccgcctggcattatgcccagtacatgaccttatgggactttcctacttg

  gcagtacatctacgtattagtcatcgctattaccatggtgatgcggtttt

  ggcagtacatcaatgggcgtggatagcggtttgactcacggggatttcca

  agtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatca

  acggttaacaagcttataacttcgtatagcatacattatacgaagttatt

  acgtagcggccgcgtcgacgatatcgctgccggagcccccggggccgctg

  ccggaagatctggcattgctgtgactgtggtgtaggccggcagctggagc

  tctgattagacccctcacctgggaatctccatatgctgcacgtgcggccc

  taaaaagacaaaagacaaaaaaaaaaaaaaaaaaaaaaaatcaaaaaaaa

  acatagggggttaccaacgtggggtccagaaagatgtggttttctcccat

  tggccttgcccagttacctatatcagtccttgtccaacaggggttttagg

  ggtggaaatgccccataaattttacggtttctttgcccttctcttccttt

  agactgagtcaccattgctctcattccttttctatcagttgaggagtggg

  ttagagattaaggtccatgtggtggaggtacacttcttatagtaaacaag

  gcctatggggaattactctctggagcccttaaaccacaaatgataatcca

  tgccacatcaaagatgcatcgaagcccatgctcctacactgactacctga

  gttagcattctgcctcaacaggactgaccatccccagctctggggcagat

  atcctctctctgccacaagggcagtgacccccatgctgtctgagggtcac

  gctttaccccccccccacccctgccgtgaccccccagaccaccccaggag

  gtgggcactaatatccctcattaccccatagatgaggaaacagaggttcc

  cccggggtcccacaggtgctcagggtcacatgcaccgtgggcacccaggc

  cccatcccaaggccaccctccctcctcaggaagctgtgctgcgctgggcc

  agaaggtactgcacacgactcctcagcctccggtggtgggaggcagcctc

  aagcctctgagtgggggggcacccgggctcctcaatctatactgactcct

  gggggtgggagaaggggagggggagctgtggcctctgagtccactaagca

  aatcagggtgggcaatgcgggcccatttcaaggaggagagaaccgaggct

  ctgacagcaggccgggggtccagggacctgcccagggtcataggctgaac

  tgctggctgacctgccttgggttctttccttggctcctcagccctgtgtg

  atgtgacaggtcattcattcactcactcgctcattcattcagcaaaccct

  cagtgagccctgctgggagcaggtgctaggggcaaggagacaggacctct

  tgccctggaacagctgaagcactgggggacaggcagtggcagggaggtgc

  gtgatcaccgctgaccccattccatcctccagcccccaggtcagtttcca

  cccaccattgaccccaccatgtcctccatccccaaggtcagtttcccgcc

  caaggagcatctccttacacactagggacaaaatttcacggctgtcactg

  ggcatctctccacgctcatcacagccctctagcagccttgaagtcctgta

  gagcccttcccatttcacagaagggacaagactatgagggccacaccgtg

  agccatgagccttaggctgtgagccgggacagcccctgcaggactggtgg

  cctcagggcactgggtggggagggtgcacagtgggtgggccccttgtgga

  atagagaggagtgtcaggtcaggggagggggcttggcctggccctggcct

  gcctggtgtgcaaccctaggcagcccctccttcccaggcctcctacttcc

  tggaggccaagcctcagggaggtaattgagtcaggtgggggagggggggt

  tgtggctttcttcacagcagaaaaacagagcccacaatagtgtccactga

  gacagaggggtcctgggggaggggaggggtgggaggtgactgctgagccc

  tgtgggagggagggagcaactactgagctgagctgggtgactctcccatc

  tgccccgccccctgtggggccagcagagtcaccgagagaacatgacccag

  ccaggcctggacagggggacacccatgtcctttaccccacagggttcact

  gagcctatctgccccaagcctgtgtctccctgggacggagaccctcactc

  ccaaccacaaaggtctaaactcaagttcccaacagccttgaaaatacagc

  ttccgggggcctccaaggagcagtcagccgtccactgccaggctcgctgg

  ctcagtgacacaggacacatcctgatgacggtccacctgtctccaagcag

  gttctcctctgccgatggggcaacgagctcctcctgtggctccctggctg

  gatgcgtgggaggcggggtgggggggcaggcggtgttcctggccgcacac

  aaggagcacccccaccagcatccgaagacgggggcccggtctttccccaa

  aacactgcttgcgggagactttgtgacgtttccaggggccatgctccctt

  cgggcagcttgggggacttctgctcctatgtggtcacctgcagggactcc

  ccccaggccttggggacaaacaaagtgatgagagggagggttagtgggtc

  ggggcagggccagtctttggaccggtttatctgaaaagccagttggtcac

  cgggaaccacagcaaacctaaacccatttggccaggcatctcccagggac

  agtctcccccaggatgcggggcccaggggggctccaggggtgacctgcgt

  cctggatttccctgatgctcccagttcgtgcctctgtccaagcatgattt

  ttaatagtgccccttccactcccagaaatgtccaagtgtgggcaataaat

  tctggtcacctgagctcagtgtaactgtttgctgaatgacacttactgta

  acaggttaaaatgggaggcccaaggccacgcagagccatcgaaggctctg

  tgtgtcccagccctgatagaagcatcaggatggggactgtggcctcacca

  ggggccacatccaggcggtcaccatggggttcctggtctccgtgggcctt

  gactggagcccctggtgtgagctcaccccatcccagcctgtgagaggcct

  ggatgtgggcctgacatcatttcccacccagtgacagcactgcatgtgat

  ggggcctctgggcagcctttttcccgggggaaactggcaggaatcaggac

  caccaggacaggggtcaggggagaggcgatgctgggcaccagagcctgga

  ccaccctcgggttctcagcgatgggcaacccctgccacccagggccccgc

  cttcctggggagacatcggggtttccaggccatcctgggaggagggtggg

  agcctcagctagaccccagctggcttgcccccccatgccccggccaagag

  agggtcttggagggaagggggaccccagaccagcctggcgagcccatcct

  cagggtctctggtcagacaggggctcagctgagctccagggtagaccaag

  gccctgcgtggatgaggccagtgtggtcactgcccagagcaaagccacct

  ctcagcagccctttcctgagcaccttctgtgtgcggggacatcagcagtg

  gcaacacagccatgctggggactcagggctagagacaggggaccagccta

  tggagagtgggtagtgtcctgcagggcaggcttgtgccctggagaaaaca

  aaccagggtgaggccagggacgctggccgggttcacagggtgatggctga

  gcacagagtgccaggggctggactgtcctgactctgggttggtggctgag

  ggcctgtgtccctctatgcctctgggttggtgataatggaaacttgctcc

  ctggagagacaggacgaatggttgatgggaaatgaatgtttgcttgtcac

  ttggttgactgttgttgccgttagcattgggcttcttgggccaggcagcc

  tcaggccagcactgctgggctccccacaggcccgacaccctcagccctgt

  gcagctggcctggcgaaaccaagaggccctgatgcccaaaatagccggga

  aaccccaaccagcccagccctggcagcaggtgcctcccatttgcctgggc

  tgggggaggggtggctctggttctggaagtttctgccagtccagctggag

  aagggacctgtatcccagcacccaggccgcccaagcccctgcaccagggc

  ctgggccaggcagagttgacatcaatcaattgggagctgctggaatgcat

  ggaggcggcgctctcgaggctggaggaggccagctgatttaaatcggtcc

  gcgtacgatgcatattaccctgttatccctaccgcggttactggccgtcg

  ttttacaacgtcgtgactgggaaaaccctggcgatgctcttctcccggtg

  aaaacctctgacacatggctcttctaaatccggagtttaaacgcttcctt

  catgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgt

  tgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaat

  cgacgctcaagtcagaggtggcgaaacccgacaggactataaagatacca

  ggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgc

  cgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctt

  tctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctc

  caagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcct

  tatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcg

  ccactggcagcagccactggtaacaggattagcagagcgaggtatgtagg

  cggtgctacagagttcttgaagtggtggcctaactacggctacactagaa

  ggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaa

  agagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtgg

  tttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaag

  aagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaac

  tcacgttaagggattttggtcatgcctaggtggcaaacagctattatggg

  tattatgggtctaccggtgcatgagattatcaaaaaggatcttcacctag

  atccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatga

  gtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct

  cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtg

  tagataactacgatacgggagggcttaccatctggccccagtgctgcaat

  gataccgcgagacccacgctcaccggctccagatttatcagcaataaacc

  agccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcc

  tccatccagtctattaattgttgccgggaagctagagtaagtagttcgcc

  agttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgt

  cacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatca

  aggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctcctt

  cggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactca

  tggttatggcagcactgcataattctcttactgtcatgccatccgtaaga

  tgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtg

  tatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccg

  cgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcg

  gggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgta

  acccactcgtgcacccaactgatcttcagcatcttttactttcaccagcg

  tttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaata

  agggcgacacggaaatgttgaatactcatactcttcctttttcaatatta

  ttgaagcatttatcagggttattgtctcgggagcggatacatatttgaat

  gtatttagaaaaa

  SEQ ID 46

  taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacg

  tcgctgagcaggccctggcctccctggccgagggcggtttgcgtattaga

  ggcctaaatggccgaattcagcggataacaatttcacacaggaaacagct

  atgaccatgattatctagtaactataacggtcctaaggtagcgagcgatc

  gcttaattaacctgcagggatatcccatgggggccgccagtgtgatggat

  atctgcagaattcgcccttgatattaagagaagggcaagtcagcttaagt

  ttgggggtagaggggaacagggagtgaggagatctggcctgagagatagg

  agccctggtggccacaggaggactctttgggtcctgtcggatggacacag

  ggcggcccgggggcatgttggagcccggctggttcttaccagaggcaggg

  ggcaccctctgacacgggagcagggcatgttccatacatgacacacccct

  ctgctccagggcaggtgggtggcggcacagaggagccagggactctgagc

  aaggggtccaccagtggggcagttggatccagacttctctgggccagcga

  gagtctagccctcagccgttctctgtccaggaggggggtggggcaggcct

  gggcggccagagctcatccctcaagggttcccagggtcctgccagaccca

  gatttccgaccgcagccaccacaagaggatgtggtctgctgtggcagctg

  ccaagaccttgcagcaggtgcagggtgggggggtgggggcacctgggggc

  agctggggtcactgagttcagggaaaaccccttttttcccctaaacctgg

  ggccatccctaggggaaaccacaacttctgagccctgggcagtggctgct

  gggagggaagagcttcatcctggaccctgggggggaacccagctccaaag

  gtgcaaggggcccaggtccaaggctagagtgggccaagcaccgcaatggc

  cagggagtgggggaggtggagctggactggatcagggcctccttgggact

  ccctacaccctgtgtgacatgttagggtacccacaccccatcaccagtca

  gggcctggcccatctccagggccagggatgtgcatgtaagtgtgtgtgag

  tgtgtgtgtgtggtgtagtacaccccttggcatccggttccgaggccttg

  ggttcctccaaagttgctctctgaattaggtcaaactgtgaggtcctgat

  cgccatcatcaacttcgttctccccacctcccatcattatcaagagctgg

  ggagggtctgggatttcttcccacccacaagccaaaagataagcctgctg

  gtgatggcagaagacacaggatcctgggtcagagacaaaggccagtgtgt

  cacagcgagagaggcagccggactatcagctgtcacagagaggccttagt

  ccgctgaactcaggccccagtgactcctgttccactgggcactggccccc

  ctccacagcgcccccaggccccagggagaggcgtcacagcttagagatgg

  ccctgctgaacagggaacaagaacaggtgtgccccatccagcgccccagg

  ggtgggacaggtgggctggatttggtgtgaagcccttgagccctggaacc

  caaccacagcagggcagttggtagatgccatttggggagaggccccagga

  gtaagggccatgggcccttgagggggccaggagctgaggacagggacaga

  gacggcccaggcagaggacagggccatgaggggtgcactgagatggccac

  tgccagcaggggcagctgccaacccgtccagggaacttattcagcagtca

  gctggaggtgccattgaccctgagggcagatgaagcccaggccaggctag

  gtgggctgtgaagaccccaggggacagagctctgtccctgggcagcactg

  gcctctcattctgcagggcttgacgggatcccaaggcctgctgcccctga

  tggtagtggcagtaccgcccagagcaggaccccagcatggaaaccccaac

  gggacgcagcctgcggagcccacaaaaccagtaaggagccgaagcagtca

  tggcacggggagtgtggacttccctttgatggggcccaggcatgaaggac

  agaatgggacagcggccatgagcagaaaatcagccggaggggatgggcct

  aggcagacgctggctttatttgaagtgttggcattttgtctggtgtgtat

  tgttggtattgattttattttagtatgtcagtgacatactgacatattat

  gtaacgacatattattatgtgttttaagaagcactccaagggaacaggct

  gtctgtaatgtgtccagagaagagagcaagagcttggctcagtctccccc

  aaggaggtcagttcctcaacaggggtcctaaatgtttcctggagccaggc

  ctgaatcaagggggtcatatctacacgtggggcagacccatggaccattt

  tcggagcaataagatggcagggaggataccaagctggtcttacagatcca

  gggctttgacctgtgacgcgggcgctcctccaggcaaagggagaagccag

  caggaagctttcagaactggggagaacagggtgcagacctccagggtctt

  gtacaacgcaccctttatcctggggtccaggaggggtcactgagggattt

  aagtgggggaccatcagaaccaggtttgtgttttggaaaaatggctccaa

  agcagagaccagtgtgaggccagattagatgatgaagaagaggcagtgga

  aagtcgatgggtggccaggtagcaagagggcctatggagttggcaagtga

  atttaaagtggtggcaccagagggcagatggggaggagcaggcactgtca

  tggactgtctatagaaatctaaaatgtataccctttttagcaatatgcag

  tgagtcataaaagaacacatatatatttcctttggccggccggcgcgcca

  cgcgtataacttcgtatagcatacattatacgaagttatcttaagggcta

  tggcagggcctgccgccccgacgttggctgcgagccctgggccttcaccc

  gaacttggggggtggggtggggaaaaggaagaaacgcgggcgtattggcc

  ccaatggggtctcggtggggtatcgacagagtgccagccctgggaccgaa

  ccccgcgtttatgaacaaacgacccaacaccgtgcgttttattctgtctt

  tttattgccgtcatagcgcgggttccttccggtattgtctccttccgtgt

  ttcactcgagttagaagaactcgtcaagaaggcgatagaaggcgatgcgc

  tgcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcagccca

  ttcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcct

  gatagcggtccgccacacccagccggccacagtcgatgaatccagaaaag

  cggccattttccaccatgatattcggcaagcaggcatcgccatgggtcac

  gacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagtt

  cggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgaca

  agaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttg

  gtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattg

  catcagccatgatggatactttctcggcaggagcaaggtgagatgacagg

  agatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttc

  agtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagcc

  acgatagccgcgctgcctcgtcctgcagttcattcagggcaccggacagg

  tcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacac

  ggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaata

  gcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttca

  atggccgatcccattccagatctgttagcctcccccatctcccgtgcaaa

  cgtgcgcgccaggtcgcagatcgtcggtatggagcctggggtggtgacgt

  gggtctggatcatcccggaggtaagttgcagcagggcgtcccggcagccg

  gcgggcgattggtcgtaatccaggataaagacgtgcatgggacggaggcg

  tttggtcaagacgtccaaggcccaggcaaacacgttgtacaggtcgccgt

  tgggggccagcaactcgggggcccgaaacagggtaaataacgtgtccccg

  atatggggtcgtgggcccgcgttgctctggggctcggcaccctggggcgg

  cacggccgtccccgaaagctgtccccaatcctcccgccacgacccgccgc

  cctgcagataccgcaccgtattggcaagcagcccgtaaacgcggcgaatc

  gcggccagcatagccaggtcaagccgctcgccggggcgctggcgtttggc

  caggcggtcgatgtgtctgtcctccggaagggcccccaacacgatgtttg

  tgccgggcaaggtcggcgggatgagggccacgaacgccagcacggcctgg

  ggggtcatgctgcccataaggtatcgcgcggccgggtagcacaggagggc

  ggcgatgggatggcggtcgaagatgagggtgagggccgggggcggggcat

  gtgagctcccagcctcccccccgatatgaggagccagaacggcgtcggtc

  acggcataaggcatgcccattgttatctgggcgcttgtcattaccaccgc

  cgcgtccccggccgatatctcaccctggtcaaggcggtgttgtgtggtgt

  agatgttcgcgattgtctcggaagcccccagcacccgccagtaagtcatc

  ggctcgggtacgtagacgatatcgtcgcgcgaacccagggccaccagcag

  ttgcgtggtggtggttttccccatcccgtggggaccgtctatataaaccc

  gcagtagcgtgggcattttctgctccgggcggacttccgtggcttcttgc

  tgccggcgagggcgcaacgccgtacgtcggttgctatggccgcgagaacg

  cgcagcctggtcgaacgcagacgcgtgctgatggccggggtacgaagcca

  tggtggctctagaggtcgaaaggcccggagatgaggaagaggagaacagc

  gcggcagacgtgcgcttttgaagcgtgcagaatgccgggcttccggagga

  ccttcgggcgcccgccccgcccctgagcccgcccctgagcccgcccccgg

  acccaccccttcccagcctctgagcccagaaagcgaaggagccaaagctg

  ctattggccgctgccccaaaggcctacccgcttccattgctcagcggtgc

  tgtccatctgcacgagactagtgagacgtgctacttccatttgtcacgtc

  ctgcacgacgcgagctgcggggcgggggggaacttcctgactaggggagg

  agtagaaggtggcgcgaaggggccaccaaagaacggagccggttggcgcc

  taccggtggatgtggaatgtgtgcgaggccagaggccacttgtgtagcgc

  caagtgcccagcggggctgctaaagcgcatgctccagactgccttgggaa

  aagcgcctcccctacccggtagggatccgcgttacataacttacggtaaa

  tggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataa

  tgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaa

  tgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgta

  tcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccg

  cctggcattatgcccagtacatgaccttatgggactttcctacttggcag

  tacatctacgtattagtcatcgctattaccatggtgatgcggttttggca

  gtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtc

  tccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgg

  ttaacaagcttagatctgcggccgcgtcgacgataaattgtgtaattcca

  cttctaaggattcatcccaaggggggaaaataatcaaagatgtaaccaaa

  ggtttacaaacaagaactcatcattaatcttccttgttgttatttcaacg

  atattattattattactattattattattattattttgtctttttgcatt

  ttctagggccactcccacggcatagagaggttcccaggctaggggtcaaa

  tcggagctacagctgccggcctacgccagagccacagcaacgcaggatct

  gagccacagcaatgcaggatctacaccacagctcatggtaacgctggatc

  cttaacccaatgagtgaggccagggatcgaacctgtaacttcatggttcc

  tagtcggattcattaaccactgagccacgacaggaactccaacattatta

  atgatgggagaaaactggaagtaacctaaatatccagcagaaagggtgtg

  gccaaatacagcatggagtagccatcataaggaatcttacacaagcctcc

  aaaattgtgtttctgaaattgggtttaaagtacgtttgcattttaaaaag

  cctgccagaaaatacagaaaaatgtctgtgatatgtctctggctgatagg

  attttgcttagttttaattttggctttataattttctatagttatgaaaa

  tgttcacaagaagatatatttcattttagcttctaaaataattataacac

  agaagtaatttgtgctttaaaaaaatattcaacacagaagtatataaagt

  aaaaattgaggagttcccatcgtggctcagtgattaacaaacccaactag

  tatccatgaggatatggatttgatccctggccttgctcagtgggttgagg

  atccagtgttgctgtgagctgtggtgtaggttgcagacacagcactctgg

  cgttgctgtgactctggcgtaggccggcagctacagctccatttggaccc

  ttagcctgggaacctccatatgcctgagatacggccctaaaaagtcaaaa

  gccaaaaaaatagtaaaaattgagtgtttctacttaccacccctgcccac

  atcttatgctaaaacccgttctccagagacaaacatcgtcaggtgggtct

  atatatttccagccctcctcctgtgtgtgtatgtccgtaaaacacacaca

  cacacacacacacgcacacacacacacacgtatctaattagcattggtat

  tagtttttcaaaagggaggtcatgctctaccttttaggcggcaaatagat

  tatttaaacaaatctgttgacattttctatatcaacccataagatctccc

  atgttcttggaaaggctttgtaagacatcaacatctgggtaaaccagcat

  ggtttttagggggttgtgtggatttttttcatattttttagggcacacct

  gcagcatatggaggttcccaggctaggggttgaatcagagctgtagctgc

  cggcctacaccacagccacagcaacgccagatccttaacccactgagaaa

  ggccagggattgaacctgcatcctcatggatgctggtcagatttatttct

  gctgagccacaacaggaactccctgaaccagaatgcttttaaccattcca

  ctttgcatggacatttagattgtttccatttaaaaatacaaattacaagg

  agttcccgtcgtggctcagtggtaacgaattggactaggaaccatgaggt

  ttcgggttcgatccctggccttgctcggtgggttaaggatccagcattga

  tgtgagatatggtgtaggtcgcagacgtggctcggatcccacgttgctgt

  ggctctggcgtaggccggcaacaacagctccgattcgacccctagcctgg

  gaacctccatgtgccacaggagcagccctagaaaaggcaaaaagacaaaa

  aaataaaaaattaaaatgaaaaaataaaataaaaatacaaattacaagag

  acggctacaaggaaatccccaagtgtgtgcaaatgccatatatgtataaa

  atgtactagtgtctcctcgcgggaaagttgcctaaaagtgggttggctgg

  acagagaggacaggctttgacattctcataggtagtagcaatgggcttct

  caaaatgctgttccagtttacactcaccatagcaaatgacagtgcctctt

  cctctccacccttgccaataatgtgacaggtggatctttttctattttgt

  gtatctgacaagcaaaaaatgagaacaggagttcctgtcgtggtgcagtg

  gagacaaatctgactaggaaccatgaaatttcgggttcaatccctggcct

  cactcagtaggtaaaggatccagggttgcagtgagctgtggggtaggtcg

  cagacacagtgcaaatttggccctgttgtggctgtggtgtaggccggcag

  ctatagctccaattggacccctagcctgggaacctccttatgccgtgggt

  gaggccctaaaaaaaagagtgcaaaaaaaaaaaataagaacaaaaatgat

  catcgtttaattctttatttgatcattggtgaaacttattttccttttat

  atttttattgactgattttatttctcctatgaatttaccggtcatagttt

  tgcctgggtgtttttactccggttttagttttggttggttgtattttctt

  agagagctatagaaactcttcatctatttggaatagtaattcctcattaa

  gtatttgtgctgcaaaaaattttccctgatctgttttatgcttttgtttg

  tggggtctttcacgagaaagcctttttagtttttacacctcagcttggtt

  gtttttcttgattgtgtctgtaatctgcggccaacataggaaacacattt

  ttactttagtgtttttttcctattttcttcaagtacgtccattgttttgg

  tgtctgattttactttgcctggggtttgtttttgtgtggcaggaatataa

  acttatgtattttccaaatggagagccaatggttgtatatttgttgaatt

  caaatgcaactttatcaaacaccaaatcatcgatttatcacaactcttct

  ctggtttattgatctaatgatcaattcctgttccacgctgttttaattat

  tttagctttgtggattttggtgcctggtagagaacaaagcctccattatt

  ttcattcaaaatagtcccgtctattatctgccattgttgtagtattagac

  tttaaaatcaatttactgattttcaaaagttattcctttggtgatgtgga

  atactttatacttcataaggtacatggattcatttgtggggaattgatgt

  ctttgctattgtggccatttgtcaagttgtgtaatattttacccatgcca

  actttgcatattgtatgtgagtttattcccagggtttttaataggatgtt

  tattgaagttgtcagtgtttccacaatttcatcgcctcagtgcttactgt

  ttgcataaaaggaaacctactcacttttgcctattgctcttgtattcaat

  cattttagttaactcttgtgttaattttgagagtttttcagctgactgtc

  tggggttttctttaatagactagccctttgtctgtaaagaataattttat

  cgaatttttcttaacactcacactctccccacccccacccccgctcatct

  cctttcattgggtcaaatctgtagaatacaataaaagtaagagtgggaac

  cttagcctttaagtcgattttgcctttaaatgtgaatgttgctatgtttc

  gggacattctctttatcaagttgcggatgtttccttagataattaactta

  ataaaagactggatgtttgctttcttcaaatcagaattgtgttgaattta

  tattgctattctgtttaattttgtttcaaaaaatttacatgcacacctta

  aagataaccatgaccaaatagtcctcctgctgagagaaaatgttggcccc

  aatgccacaggttacctcccgactcagataaactacaatgggagataaaa

  tcagatttggcaaagcctgtggattcttgccataactctcagagcatgac

  ttgggtgttttttccttttctaagtattttaatggtatttttgtgttaca

  ataggaaatctaggacacagagagtgattcaatgaggggaacgcattctg

  ggatgactctaggcctctggtttggggagagctctattgaagtaaagaca

  atgagaggaagcaagtttgcagggaactgtgaggaatttagatggggaat

  gttgggtttgaggtttctatagggcacgcaagcagagatgcactcaggag

  gaagaaggagcataaatctagtggcgctgccggcaagcttgctggaggag

  gccaattgggagctgctggaatgcatggaggcggcgctctcgaggctgga

  ggaggccagctgatttaaatcggtccgcgtacgatgcatattaccctgtt

  atccctaccgcggttactggccgtcgttttacaacgtcgtgactgggaaa

  accctggcgatgctcttctcccggtgaaaacctctgacacatggctcttc

  taaatccggagtttaaacgcttccttcatgtgagcaaaaggccagcaaaa

  ggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctcc

  gcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcga

  aacccgacaggactataaagataccaggcgtttccccctggaagctccct

  cgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcct

  ttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtat

  ctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacc

  ccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagt

  ccaacccggtaagacacgacttatcgccactggcagcagccactggtaac

  aggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtg

  gtggcctaactacggctacactagaaggacagtatttggtatctgcgctc

  tgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggc

  aaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagat

  tacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg

  ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatg

  cctaggtggcaaacagctattatgggtattatgggtctaccggtgcatga

  gattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagt

  tttaaatcaatctaaagtatatatgagtaaacttggtctgacagttacca

  atgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcat

  ccatagttgcctgactccccgtcgtgtagataactacgatacgggagggc

  ttaccatctggccccagtgctgcaatgataccgcgagacccacgctcacc

  ggctccagatttatcagcaataaaccagccagccggaagggccgagcgca

  gaagtggtcctgcaactttatccgcctccatccagtctattaattgttgc

  cgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgt

  tgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggctt

  cattcagctccggttcccaacgatcaaggcgagttacatgatcccccatg

  ttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaag

  taagttggccgcagtgttatcactcatggttatggcagcactgcataatt

  ctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtac

  tcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttg

  cccggcgtcaatacgggataataccgcgccacatagcagaactttaaaag

  tgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatctta

  ccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatc

  ttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaa

  ggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaata

  ctcatactcttcctttttcaatattattgaagcatttatcagggttattg

  tctcgggagcggatacatatttgaatgtatttagaaaaa

  SEQ ID 47

  taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacg

  tcgctgagcaggccctggcctccctggccgagggcggtttgcgtattaga

  ggcctaaatggccgaattcagcggataacaatttcacacaggaaacagct

  atgaccatgattatctagtaactataacggtcctaaggtagcgagcgatc

  gcttaattaacctgcagggataaccactgacccatgacgggaactcccag

  ggctcagctcttgactccaggttcgcagctgccctcaaagcaatgcaacc

  ctggctggccccgcctcatgcatccggcctcctccccaaagagctctgag

  cccacctgggcctaggtcctcctccctgggactcatggcctaagggtaca

  gagttactggggctgatgaagggaccaatggggacaggggcctcaaatca

  aagtggctgtctctctcatgtcccttcctctcctcagggtccaaaatcag

  ggtcagggccccagggcaggggctgagagggcctctttctgaaggccctg

  tctcagtgcaggttatgggggtctgggggagggtcaatgcagggctcacc

  cttcagtgccccaaagcctagagagtgagtgcctgccagtggcttcccag

  gcccaatcccttgactgcctgggaatgctcaaatgcaggaactgtcacaa

  caccttcagtcaggggctgctctgggaggaaaaacactcagaattggggg

  ttcagggaaggcccagtgccaagcatagcaggagctcaggtggctgcaga

  tggtgtgaaccccaggagcaggatggccggcactccccccagaccctcca

  gagccccaggttggctgccctcttcactgccgacacccctgggtccactt

  ctgccctttcccacctaaaacctttagggctcccactttctcccaaatgt

  gagacatcaccacggctcccagggagtgtccagaagggcatctggctgag

  aggtcctgacatctgggagcctcaggccccacaatggacagacgccctgc

  caggatgctgctgcagggctgttagctaggcggggtggagatggggtact

  ttgcctctcagaggccccggccccaccatgaaacctcagtgacaccccat

  ttccctgagttcacatacctgtatcctactccagtcaccttccccacgaa

  cccctgggagcccaggatgatgctggggctggagccacgaccagcccacg

  agtgatccagctctgccaatcagcagtcatttcccaagtgttccagccct

  gccaggtcccactacagcagtaatggaggccccagacaccagtccagcag

  ttagagggctggactagcaccagctttcaagcctcagcatctcaaggtga

  atggccagtgcccctccccgtggccatcacaggatcgcagatatgaccct

  aggggaagaaatatcctgggagtaaggaagtgcccatactcaaggatggc

  ccctctgtgacctaacctgtccctgaggattgtacttccaggcgttaaaa

  cagtagaacgcctgcctgtgaacccccgccaagggactgcttggggaggc

  cccctaaaccagaacacaggcactccagcaggacctctgaactctgacca

  ccctcagcaagtgggcaccccccgcagcttccaaggcaccccagggctca

  ccacagcggcccctcctggcagcccctcacccaggcccagaccctctaag

  atggcacatctaagccaatccacctccttgtcattcctcctgtccccacc

  caggacccttctcagatgaaaccttcgctccagccgctgggccctctctc

  ctgcccctctggcagttctccagggactccgcctcccactctctgtctct

  ccctgcactcctaggaacaagcgacctccaggaagcccagtccaattatc

  ccctctgtgtcctccccaatctctgcctctgggtggatttgagcaccaca

  tcctgttctcttcgacctgaaactccttggccccggtgtccgctctcctg

  ggccctcttttctctcctcccctcttccgtgccccgtttgtttggtgtta

  caggcaggccccggggagccgtccctccagctgctcttccttgtctgtct

  caggagccagaaactggcagcatctaaaaagggctcctgtttcttcatct

  gcccagcctcctagcccaaccagggctctggcctcactccagagggtggg

  ctccagagggcaggggttgcaccctcttagtgcctcagaggctcagctgg

  gtgcaggatgggggggccctcagggagcccctcagtgactgctgatcact

  tactgcaggactgttcccagctcttcccaatcattggaatgacaatacct

  agttctgctccatcatagtgatgcaggaaaaatgttactgaaatcctggt

  tcttgtttagcaatcgaagaatgaattccgcgaacacacaggcagcaagc

  aagcgaagcctttattaaaggaaagcagatagctcccagggctgcaggga

  gcggggagaagagctccccactctctattgtcctatagggctttttaccc

  cttaaagttggggggatacaaaaaaaatagaagaaaaagggagttcccgt

  cagggcacagcagaaacaaatccaactaggaaccatgaggttgggggttc

  gattcctggcctctctcagtgggttaaggatgcagcgttgccgtgagcta

  tgatacaggtcacagatgcagctcagatctactagtcaattgacaggcgc

  cggagcaggagctaggcctttggccggccggcgcgccacgcgtataactt

  cgtatagcatacattatacgaagttatcttaagggctatggcagggcctg

  ccgccccgacgttggctgcgagccctgggccttcacccgaacttgggggg

  tggggtggggaaaaggaagaaacgcgggcgtattggccccaatggggtct

  cggtggggtatcgacagagtgccagccctgggaccgaaccccgcgtttat

  gaacaaacgacccaacaccgtgcgttttattctgtctttttattgccgtc

  atagcgcgggttccttccggtattgtctccttccgtgtttcactcgagtt

  agaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcggga

  gcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaag

  ctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccg

  ccacacccagccggccacagtcgatgaatccagaaaagcggccattttcc

  accatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctc

  gccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcga

  gcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttcc

  atccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgg

  gcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatga

  tggatactttctcggcaggagcaaggtgagatgacaggagatcctgcccc

  ggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtc

  gagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcg

  ctgcctcgtcctgcagttcattcagggcaccggacaggtcggtcttgaca

  aaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcaga

  gcagccgattgtctgttgtgcccagtcatagccgaatagcctctccaccc

  aagcggccggagaacctgcgtgcaatccatcttgttcaatggccgatccc

  attccagatctgttagcctcccccatctcccgtgcaaacgtgcgcgccag

  gtcgcagatcgtcggtatggagcctggggtggtgacgtgggtctggatca

  tcccggaggtaagttgcagcagggcgtcccggcagccggcgggcgattgg

  tcgtaatccaggataaagacgtgcatgggacggaggcgtttggtcaagac

  gtccaaggcccaggcaaacacgttgtacaggtcgccgttgggggccagca

  actcgggggcccgaaacagggtaaataacgtgtccccgatatggggtcgt

  gggcccgcgttgctctggggctcggcaccctggggcggcacggccgtccc

  cgaaagctgtccccaatcctcccgccacgacccgccgccctgcagatacc

  gcaccgtattggcaagcagcccgtaaacgcggcgaatcgcggccagcata

  gccaggtcaagccgctcgccggggcgctggcgtttggccaggcggtcgat

  gtgtctgtcctccggaagggcccccaacacgatgtttgtgccgggcaagg

  tcggcgggatgagggccacgaacgccagcacggcctggggggtcatgctg

  cccataaggtatcgcgcggccgggtagcacaggagggcggcgatgggatg

  gcggtcgaagatgagggtgagggccgggggcggggcatgtgagctcccag

  cctcccccccgatatgaggagccagaacggcgtcggtcacggcataaggc

  atgcccattgttatctgggcgcttgtcattaccaccgccgcgtccccggc

  cgatatctcaccctggtcaaggcggtgttgtgtggtgtagatgttcgcga

  ttgtctcggaagcccccagcacccgccagtaagtcatcggctcgggtacg

  tagacgatatcgtcgcgcgaacccagggccaccagcagttgcgtggtggt

  ggttttccccatcccgtggggaccgtctatataaacccgcagtagcgtgg

  gcattttctgctccgggcggacttccgtggcttcttgctgccggcgaggg

  cgcaacgccgtacgtcggttgctatggccgcgagaacgcgcagcctggtc

  gaacgcagacgcgtgctgatggccggggtacgaagccatggtggctctag

  aggtcgaaaggcccggagatgaggaagaggagaacagcgcggcagacgtg

  cgcttttgaagcgtgcagaatgccgggcttccggaggaccttcgggcgcc

  cgccccgcccctgagcccgcccctgagcccgcccccggacccaccccttc

  ccagcctctgagcccagaaagcgaaggagccaaagctgctattggccgct

  gccccaaaggcctacccgcttccattgctcagcggtgctgtccatctgca

  cgagactagtgagacgtgctacttccatttgtcacgtcctgcacgacgcg

  agctgcggggcgggggggaacttcctgactaggggaggagtagaaggtgg

  cgcgaaggggccaccaaagaacggagccggttggcgcctaccggtggatg

  tggaatgtgtgcgaggccagaggccacttgtgtagcgccaagtgcccagc

  ggggctgctaaagcgcatgctccagactgccttgggaaaagcgcctcccc

  tacccggtagggatccgcgttacataacttacggtaaatggcccgcctgg

  ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttc

  ccatagtaacgccaatagggactttccattgacgtcaatgggtggagtat

  ttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaag

  tacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatg

  cccagtacatgaccttatgggactttcctacttggcagtacatctacgta

  ttagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgg

  gcgtggatagcggtttgactcacggggatttccaagtctccaccccattg

  acgtcaatgggagtttgttttggcaccaaaatcaacggttaacaagctta

  taacttcgtatagcatacattatacgaagttattacgtagcggccgcgtc

  gacgatatcgctgccggagcccccggggccgctgccggaagatctggcat

  tgctgtgactgtggtgtaggccggcagctggagctctgattagacccctc

  acctgggaatctccatatgctgcacgtgcggccctaaaaagacaaaagac

  aaaaaaaaaaaaaaaaaaaaaaaatcaaaaaaaaacatagggggttacca

  acgtggggtccagaaagatgtggttttctcccattggccttgcccagtta

  cctatatcagtccttgtccaacaggggttttaggggtggaaatgccccat

  aaattttacggtttctttgcccttctcttcctttagactgagtcaccatt

  gctctcattccttttctatcagttgaggagtgggttagagattaaggtcc

  atgtggtggaggtacacttcttatagtaaacaaggcctatggggaattac

  tctctggagcccttaaaccacaaatgataatccatgccacatcaaagatg

  catcgaagcccatgctcctacactgactacctgagttagcattctgcctc

  aacaggactgaccatccccagctctggggcagatatcctctctctgccac

  aagggcagtgacccccatgctgtctgagggtcacgctttacccccccccc

  acccctgccgtgaccccccagaccaccccaggaggtgggcactaatatcc

  ctcattaccccatagatgaggaaacagaggttcccccggggtcccacagg

  tgctcagggtcacatgcaccgtgggcacccaggccccatcccaaggccac

  cctccctcctcaggaagctgtgctgcgctgggccagaaggtactgcacac

  gactcctcagcctccggtggtgggaggcagcctcaagcctctgagtgggg

  gggcacccgggctcctcaatctatactgactcctgggggtgggagaaggg

  gagggggagctgtggcctctgagtccactaagcaaatcagggtgggcaat

  gcgggcccatttcaaggaggagagaaccgaggctctgacagcaggccggg

  ggtccagggacctgcccagggtcataggctgaactgctggctgacctgcc

  ttgggttctttccttggctcctcagccctgtgtgatgtgacaggtcattc

  attcactcactcgctcattcattcagcaaaccctcagtgagccctgctgg

  gagcaggtgctaggggcaaggagacaggacctcttgccctggaacagctg

  aagcactgggggacaggcagtggcagggaggtgcgtgatcaccgctgacc

  ccattccatcctccagcccccaggtcagtttccacccaccattgacccca

  ccatgtcctccatccccaaggtcagtttcccgcccaaggagcatctcctt

  acacactagggacaaaatttcacggctgtcactgggcatctctccacgct

  catcacagccctctagcagccttgaagtcctgtagagcccttcccatttc

  acagaagggacaagactatgagggccacaccgtgagccatgagccttagg

  ctgtgagccgggacagcccctgcaggactggtggcctcagggcactgggt

  ggggagggtgcacagtgggtgggccccttgtggaatagagaggagtgtca

  ggtcaggggagggggcttggcctggccctggcctgcctggtgtgcaaccc

  taggcagcccctccttcccaggcctcctacttcctggaggccaagcctca

  gggaggtaattgagtcaggtgggggagggggggttgtggctttcttcaca

  gcagaaaaacagagcccacaatagtgtccactgagacagaggggtcctgg

  gggaggggaggggtgggaggtgactgctgagccctgtgggagggagggag

  caactactgagctgagctgggtgactctcccatctgccccgccccctgtg

  gggccagcagagtcaccgagagaacatgacccagccaggcctggacaggg

  ggacacccatgtcctttaccccacagggttcactgagcctatctgcccca

  agcctgtgtctccctgggacggagaccctcactcccaaccacaaaggtct

  aaactcaagttcccaacagccttgaaaatacagcttccgggggcctccaa

  ggagcagtcagccgtccactgccaggctcgctggctcagtgacacaggac

  acatcctgatgacggtccacctgtctccaagcaggttctcctctgccgat

  ggggcaacgagctcctcctgtggctccctggctggatgcgtgggaggcgg

  ggtgggggggcaggcggtgttcctggccgcacacaaggagcacccccacc

  agcatccgaagacgggggcccggtctttccccaaaacactgcttgcggga

  gactttgtgacgtttccaggggccatgctcccttcgggcagcttggggga

  cttctgctcctatgtggtcacctgcagggactccccccaggccttgggga

  caaacaaagtgatgagagggagggttagtgggtcggggcagggccagtct

  ttggaccggtttatctgaaaagccagttggtcaccgggaaccacagcaaa

  cctaaacccatttggccaggcatctcccagggacagtctcccccaggatg

  cggggcccaggggggctccaggggtgacctgcgtcctggatttccctgat

  gctcccagttcgtgcctctgtccaagcatgatttttaatagtgccccttc

  cactcccagaaatgtccaagtgtgggcaataaattctggtcacctgagct

  cagtgtaactgtttgctgaatgacacttactgtaacaggttaaaatggga

  ggcccaaggccacgcagagccatcgaaggctctgtgtgtcccagccctga

  tagaagcatcaggatggggactgtggcctcaccaggggccacatccaggc

  ggtcaccatggggttcctggtctccgtgggccttgactggagcccctggt

  gtgagctcaccccatcccagcctgtgagaggcctggatgtgggcctgaca

  tcatttcccacccagtgacagcactgcatgtgatggggcctctgggcagc

  ctttttcccgggggaaactggcaggaatcaggaccaccaggacaggggtc

  aggggagaggcgatgctgggcaccagagcctggaccaccctcgggttctc

  agcgatgggcaacccctgccacccagggccccgccttcctggggagacat

  cggggtttccaggccatcctgggaggagggtgggagcctcagctagaccc

  cagctggcttgcccccccatgccccggccaagagagggtcttggagggaa

  gggggaccccagaccagcctggcgagcccatcctcagggtctctggtcag

  acaggggctcagctgagctccagggtagaccaaggccctgcgtggatgag

  gccagtgtggtcactgcccagagcaaagccacctctcagcagccctttcc

  tgagcaccttctgtgtgcggggacatcagcagtggcaacacagccatgct

  ggggactcagggctagagacaggggaccagcctatggagagtgggtagtg

  tcctgcagggcaggcttgtgccctggagaaaacaaaccagggtgaggcca

  gggacgctggccgggttcacagggtgatggctgagcacagagtgccaggg

  gctggactgtcctgactctgggttggtggctgagggcctgtgtccctcta

  tgcctctgggttggtgataatggaaacttgctccctggagagacaggacg

  aatggttgatgggaaatgaatgtttgcttgtcacttggttgactgttgtt

  gccgttagcattgggcttcttgggccaggcagcctcaggccagcactgct

  gggctccccacaggcccgacaccctcagccctgtgcagctggcctggcga

  aaccaagaggccctgatgcccaaaatagccgggaaaccccaaccagccca

  gccctggcagcaggtgcctcccatttgcctgggctgggggaggggtggct

  ctggttctggaagtttctgccagtccagctggagaagggacctgtatccc

  agcacccaggccgcccaagcccctgcaccagggcctgggccaggcagagt

  tgacatcaatcaattgggagctgctggaatgcatggaggcggcgctctcg

  aggctggaggaggccagctgatttaaatcggtccgcgtacgatgcatatt

  accctgttatccctaccgcggttactggccgtcgttttacaacgtcgtga

  ctgggaaaaccctggcgatgctcttctcccggtgaaaacctctgacacat

  ggctcttctaaatccggagtttaaacgcttccttcatgtgagcaaaaggc

  cagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcca

  taggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcaga

  ggtggcgaaacccgacaggactataaagataccaggcgtttccccctgga

  agctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacct

  gtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgct

  gtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtg

  cacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcg

  tcttgagtccaacccggtaagacacgacttatcgccactggcagcagcca

  ctggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttc

  ttgaagtggtggcctaactacggctacactagaaggacagtatttggtat

  ctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctctt

  gatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaag

  cagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctt

  ttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattt

  tggtcatgcctaggtggcaaacagctattatgggtattatgggtctaccg

  gtgcatgagattatcaaaaaggatcttcacctagatccttttaaattaaa

  aatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgac

  agttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatt

  tcgttcatccatagttgcctgactccccgtcgtgtagataactacgatac

  gggagggcttaccatctggccccagtgctgcaatgataccgcgagaccca

  cgctcaccggctccagatttatcagcaataaaccagccagccggaagggc

  cgagcgcagaagtggtcctgcaactttatccgcctccatccagtctatta

  attgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgc

  aacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttgg

  tatggcttcattcagctccggttcccaacgatcaaggcgagttacatgat

  cccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgtt

  gtcagaagtaagttggccgcagtgttatcactcatggttatggcagcact

  gcataattctcttactgtcatgccatccgtaagatgcttttctgtgactg

  gtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagt

  tgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaac

  tttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaa

  ggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcaccc

  aactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaa

  aacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaat

  gttgaatactcatactcttcctttttcaatattattgaagcatttatcag

  ggttattgtctcgggagcggatacatatttgaatgtatttagaaaaa

• The two-step strategy outline above, utilizing a vector pair, can be used to delete the entire J/C cluster region (i.e., all J/C units), multiple J/C units or an individual J/C unit.
• Selectable Marker Genes
• The DNA constructs can be designed to modify the endogenous, target immunoglobulin gene. The homologous sequence for targeting the construct can have one or more deletions, insertions, substitutions or combinations thereof. The alteration can be the insertion of a selectable marker gene fused in reading frame with the upstream sequence of the target gene.
• Suitable selectable marker genes include, but are not limited to: genes conferring the ability to grow on certain media substrates, such as the tk gene (thymidine kinase) or the hprt gene (hypoxanthine phosphoribosyltransferase) which confer the ability to grow on HAT medium (hypoxanthine, aminopterin and thymidine); the bacterial gpt gene (guanine/xanthine phosphoribosyltransferase) which allows growth on MAX medium (mycophenolic acid, adenine, and xanthine). See, for example, Song, K-Y., et al. Proc. Nat'l Acad. Sci. U.S.A. 84:6820-6824 (1987); Sambrook, J., et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), Chapter 16. Other examples of selectable markers include: genes conferring resistance to compounds such as antibiotics, genes conferring the ability to grow on selected substrates, genes encoding proteins that produce detectable signals such as luminescence, such as green fluorescent protein, enhanced green fluorescent protein (eGFP). A wide variety of such markers are known and available, including, for example, antibiotic resistance genes such as the neomycin resistance gene (neo) (Southern, P., and P. Berg, J. Mol. Appl. Genet. 1:327-341 (1982)); and the hygromycin resistance gene (hyg) (Nucleic Acids Research 11:6895-6911 (1983), and Te Riele, H., et al., Nature 348:649-651 (1990)). Other selectable marker genes include: acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline.
• Methods for the incorporation of antibiotic resistance genes and negative selection factors will be familiar to those of ordinary skill in the art (see, e.g., WO 99/15650; U.S. Pat. No. 6,080,576; U.S. Pat. No. 6,136,566; Niwa et al., J. Biochem. 113:343-349 (1993); and Yoshida et al., Transgenic Research 4:277-287 (1995)).
• Combinations of selectable markers can also be used. For example, to target an immunoglobulin gene, a neo gene (with or without its own promoter, as discussed above) can be cloned into a DNA sequence which is homologous to the immunoglobulin gene. To use a combination of markers, the HSV-tk gene can be cloned such that it is outside of the targeting DNA (another selectable marker could be placed on the opposite flank, if desired). After introducing the DNA construct into the cells to be targeted, the cells can be selected on the appropriate antibiotics. In this particular example, those cells which are resistant to G418 and gancyclovir are most likely to have arisen by homologous recombination in which the neo gene has been recombined into the immunoglobulin gene but the tk gene has been lost because it was located outside the region of the double crossover.
• Deletions can be at least about 50 bp, more usually at least about 100 bp, and generally not more than about 20 kbp, where the deletion can normally include at least a portion of the coding region including a portion of or one or more exons, a portion of or one or more introns, and can or can not include a portion of the flanking non-coding regions, particularly the 5′-non-coding region (transcriptional regulatory region). Thus, the homologous region can extend beyond the coding region into the 5′-non-coding region or alternatively into the 3′-non-coding region. Insertions can generally not exceed 10 kbp, usually not exceed 5 kbp, generally being at least 50 bp, more usually at least 200 bp.
• The region(s) of homology can include mutations, where mutations can further inactivate the target gene, in providing for a frame shift, or changing a key amino acid, or the mutation can correct a dysfunctional allele, etc. The mutation can be a subtle change, not exceeding about 5% of the homologous flanking sequences. Where mutation of a gene is desired, the marker gene can be inserted into an intron or an exon.
• The construct can be prepared in accordance with methods known in the art, various fragments can be brought together, introduced into appropriate vectors, cloned, analyzed and then manipulated further until the desired construct has been achieved. Various modifications can be made to the sequence, to allow for restriction analysis, excision, identification of probes, etc. Silent mutations can be introduced, as desired. At various stages, restriction analysis, sequencing, amplification with the polymerase chain reaction, primer repair, in vitro mutagenesis, etc. can be employed.
• The construct can be prepared using a bacterial vector, including a prokaryotic replication system, e.g. an origin recognizable by E. coli, at each stage the construct can be cloned and analyzed. A marker, the same as or different from the marker to be used for insertion, can be employed, which can be removed prior to introduction into the target cell. Once the vector containing the construct has been completed, it can be further manipulated, such as by deletion of the bacterial sequences, linearization, introducing a short deletion in the homologous sequence. After final manipulation, the construct can be introduced into the cell.
• The present invention further includes recombinant constructs containing sequences of immunoglobulin genes. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. The construct can also include regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pBs, pQE-9 (Qiagen), phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSv2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPv, pMSG, pSVL (Pharmacia), viral origin vectors (M13 vectors, bacterial phage 1 vectors, adenovirus vectors, and retrovirus vectors), high, low and adjustable copy number vectors, vectors which have compatible replicons for use in combination in a single host (pACYC184 and pBR322) and eukaryotic episomal replication vectors (pCDM8). Other vectors include prokaryotic expression vectors such as pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA, B, and C, pRSET A, B, and C (Invitrogen, Corp.), pGEMEX-1, and pGEMEX-2 (Promega, Inc.), the pET vectors (Novagen, Inc.), pTrc99A, pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia, Inc.), pKK233-2 and pKK388-1 (Clontech, Inc.), and pProEx-HT (Invitrogen, Corp.) and variants and derivatives thereof. Other vectors include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), p3′SS, pXT1, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.) and variants or derivatives thereof. Additional vectors that can be used include: pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificial chromosomes), BAC's (bacterial artificial chromosomes), P1 (Escherichia coli phage), pQE70, pQE60, pQE9 (quagan), pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 and pSV-SPORT1 (Invitrogen), pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1(−)/Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZ□, pGAPZ, pGAPZ□, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SP1), pVgRXR, pcDNA2.1, pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1/Amp, pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/RSV, pREP4, pREP7, pREP8, pREP9, pREP 10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac from Invitrogen; □ ExCell, □gt11, pTrc99A, pKK223-3, pGEX-1□T, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia; pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4abc(+), pOCUS-2, pTAg, pET-32LIC, pET-30LIC, pBAC-2cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC, pT7Blue-2, □SCREEN-1, □BlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb, pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3cp, pBACgus-2cp, pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg, and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6×His-GFP, pSEAP2-Basic, pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, p□gal-Basic, p□gal-Control, p□gal-Promoter, p□gal-Enhancer, pCMV□, pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1/2/3, pYEX-S1, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTrip1Ex, □gt10, □gt11, pWE15, and □TriplEX from Clontech; Lambda ZAP II, pBK-CMV, pBK-RSV, pBluescript II KS+/−, pBluescript II SK+/−, pAD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct, pBS+/−, pBC KS+/−, pBC SK+/−, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI/MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44, pOG45, pFRT□GAL, pNEO□GAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, and pRS416 from Stratagene and variants or derivatives thereof. Two-hybrid and reverse two-hybrid vectors can also be used, for example, pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and variants or derivatives thereof. Any other plasmids and vectors may be used as long as they are replicable and viable in the host.
• Techniques which can be used to allow the DNA construct entry into the host cell include, for example, calcium phosphate/DNA co precipitation, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, or any other technique known by one skilled in the art. The DNA can be single or double stranded, linear or circular, relaxed or supercoiled DNA. For various techniques for transfecting mammalian cells, see, for example, Keown et al., Methods in Enzymology Vol. 185, pp. 527-537 (1990).
• In one specific embodiment, heterozygous or homozygous knockout cells can be produced by transfection of primary fetal fibroblasts with a knockout vector containing immunoglobulin gene sequence isolated from isogenic DNA. In another embodiment, the vector can incorporate a promoter trap strategy, using, for example, IRES (internal ribosome entry site) to initiate translation of the Neor gene.
• Site Specific Recombinases
• In additional embodiments, the targeting constructs can contain site specific recombinase sites, such as, for example, lox. In one embodiment, the targeting arms can insert the site specific recombinase target sites into the targeted region such that one site specific recombinase target site is located 5′ to the second site specific recombinase target site. Then, the site specific recombinase can be activated and/or applied to the cell such that the intervening nucleotide sequence between the two site specific recombinase sites is excised.
• Site-specific recombinases include enzymes or recombinases that recognize and bind to a short nucleic acid site or sequence-specific recombinase target site, i.e., a recombinase recognition site, and catalyze the recombination of nucleic acid in relation to these sites. These enzymes include recombinases, transposases and integrases. Examples of sequence-specific recombinase target sites include, but are not limited to, lox sites, att sites, dif sites and frt sites. Non-limiting examples of site-specific recombinases include, but are not limited to, bacteriophage P1 Cre recombinase, yeast FLP recombinase, Inti integrase, bacteriophage λ, phi 80, P22, P2, 186, and P4 recombinase, Tn3 resolvase, the Hin recombinase, and the Cin recombinase, E. coli xerC and xerD recombinases, Bacillus thuringiensis recombinase, TpnI and the β-lactamase transposons, and the immunoglobulin recombinases.
• In one embodiment, the recombination site can be a lox site that is recognized by the Cre recombinase of bacteriophage P1. Lox sites refer to a nucleotide sequence at which the product of the cre gene of bacteriophage P1, the Cre recombinase, can catalyze a site-specific recombination event. A variety of lox sites are known in the art, including the naturally occurring loxP, loxB, loxL and loxR, as well as a number of mutant, or variant, lox sites, such as loxP511, loxP514, lox.DELTA.86, lox.DELTA.117, loxC2, loxP2, loxP3 and lox P23. Additional example of lox sites include, but are not limited to, loxB, loxL, loxR, loxP, loxP3, loxP23, loxΔ86, loxΔ117, loxP511, and loxC2.
• In another embodiment, the recombination site is a recombination site that is recognized by a recombinases other than Cre. In one embodiment, the recombinase site can be the FRT sites recognized by FLP recombinase of the 2 pi plasmid of Saccharomyces cerevisiae. FRT sites refer to a nucleotide sequence at which the product of the FLP gene of the yeast 2 micron plasmid, FLP recombinase, can catalyze site-specific recombination. Additional examples of the non-Cre recombinases include, but are not limited to, site-specific recombinases include: att sites recognized by the Int recombinase of bacteriophage λ (e.g. att1, att2, att3, attP, attB, attL, and attR), the recombination sites recognized by the resolvase family, and the recombination site recognized by transposase of Bacillus thuringiensis.
• In particular embodiments of the present invention, the targeting constructs can contain: sequence homologous to a porcine immunoglobulin gene as described herein, a selectable marker gene and/or a site specific recombinase target site.
• Selection of Homologously Recombined Cells
• The cells can then be grown in appropriately-selected medium to identify cells providing the appropriate integration. The presence of the selectable marker gene inserted into the immunoglobulin gene establishes the integration of the target construct into the host genome. Those cells which show the desired phenotype can then be further analyzed by restriction analysis, electrophoresis, Southern analysis, polymerase chain reaction, etc to analyze the DNA in order to establish whether homologous or non-homologous recombination occurred. This can be determined by employing probes for the insert and then sequencing the 5′ and 3′ regions flanking the insert for the presence of the immunoglobulin gene extending beyond the flanking regions of the construct or identifying the presence of a deletion, when such deletion is introduced. Primers can also be used which are complementary to a sequence within the construct and complementary to a sequence outside the construct and at the target locus. In this way, one can only obtain DNA duplexes having both of the primers present in the complementary chains if homologous recombination has occurred. By demonstrating the presence of the primer sequences or the expected size sequence, the occurrence of homologous recombination is supported.
• The polymerase chain reaction used for screening homologous recombination events is known in the art, see, for example, Kim and Smithies, Nucleic Acids Res. 16:8887-8903, 1988; and Joyner et al., Nature 338:153-156, 1989. The specific combination of a mutant polyoma enhancer and a thymidine kinase promoter to drive the neomycin gene has been shown to be active in both embryonic stem cells and EC cells by Thomas and Capecchi, supra, 1987; Nicholas and Berg (1983) in Teratocarcinoma Stem Cell, eds. Siver, Martin and Strikland (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. (pp. 469-497); and Linney and Donerly, Cell 35:693-699, 1983.
• The cell lines obtained from the first round of targeting are likely to be heterozygous for the targeted allele. Homozygosity, in which both alleles are modified, can be achieved in a number of ways. One approach is to grow up a number of cells in which one copy has been modified and then to subject these cells to another round of targeting using a different selectable marker. Alternatively, homozygotes can be obtained by breeding animals heterozygous for the modified allele, according to traditional Mendelian genetics. In some situations, it can be desirable to have two different modified alleles. This can be achieved by successive rounds of gene targeting or by breeding heterozygotes, each of which carries one of the desired modified alleles.
• Identification of Cells that have Undergone Homologous Recombination
• In one embodiment, the selection method can detect the depletion of the immunoglobulin gene directly, whether due to targeted knockout of the immunoglobulin gene by homologous recombination, or a mutation in the gene that results in a nonfunctioning or nonexpressed immunoglobulin. Selection via antibiotic resistance has been used most commonly for screening (see above). This method can detect the presence of the resistance gene on the targeting vector, but does not directly indicate whether integration was a targeted recombination event or a random integration. Certain technology, such as Poly A and promoter trap technology, increase the probability of targeted events, but again, do not give direct evidence that the desired phenotype, a cell deficient in immunoglobulin gene expression, has been achieved. In addition, negative forms of selection can be used to select for targeted integration; in these cases, the gene for a factor lethal to the cells is inserted in such a way that only targeted events allow the cell to avoid death. Cells selected by these methods can then be assayed for gene disruption, vector integration and, finally, immunoglobulin gene depletion. In these cases, since the selection is based on detection of targeting vector integration and not at the altered phenotype, only targeted knockouts, not point mutations, gene rearrangements or truncations or other such modifications can be detected.
• Animal cells believed to lacking expression of functional immunoglobulin genes can be further characterized. Such characterization can be accomplished by the following techniques, including, but not limited to: PCR analysis, Southern blot analysis, Northern blot analysis, specific lectin binding assays, and/or sequencing analysis.
• PCR analysis as described in the art can be used to determine the integration of targeting vectors. In one embodiment, amplimers can originate in the antibiotic resistance gene and extend into a region outside the vector sequence. Southern analysis can also be used to characterize gross modifications in the locus, such as the integration of a targeting vector into the immunoglobulin locus. Whereas, Northern analysis can be used to characterize the transcript produced from each of the alleles.
• Further, sequencing analysis of the cDNA produced from the RNA transcript can also be used to determine the precise location of any mutations in the immunoglobulin allele.
• In another aspect of the present invention, ungulate cells lacking at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the process, sequences and/or constructs described herein are provided. These cells can be obtained as a result of homologous recombination. Particularly, by inactivating at least one allele of an ungulate heavy chain, kappa light chain or lambda light chain gene, cells can be produced which have reduced capability for expression of porcine antibodies. In other embodiments, mammalian cells lacking both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be produced according to the process, sequences and/or constructs described herein. In a further embodiment, porcine animals are provided in which at least one allele of an ungulate heavy chain, kappa light chain and/or lambda light chain gene is inactivated via a genetic targeting event produced according to the process, sequences and/or constructs described herein. In another aspect of the present invention, porcine animals are provided in which both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene are inactivated via a genetic targeting event. The gene can be targeted via homologous recombination. In other embodiments, the gene can be disrupted, i.e. a portion of the genetic code can be altered, thereby affecting transcription and/or translation of that segment of the gene. For example, disruption of a gene can occur through substitution, deletion (“knock-out”) or insertion (“knock-in”) techniques. Additional genes for a desired protein or regulatory sequence that modulate transcription of an existing sequence can be inserted.
• In embodiments of the present invention, alleles of ungulate heavy chain, kappa light chain or lambda light chain gene are rendered inactive according to the process, sequences and/or constructs described herein, such that functional ungulate immunoglobulins can no longer be produced. In one embodiment, the targeted immunoglobulin gene can be transcribed into RNA, but not translated into protein. In another embodiment, the targeted immunoglobulin gene can be transcribed in an inactive truncated form. Such a truncated RNA may either not be translated or can be translated into a nonfunctional protein. In an alternative embodiment, the targeted immunoglobulin gene can be inactivated in such a way that no transcription of the gene occurs. In a further embodiment, the targeted immunoglobulin gene can be transcribed and then translated into a nonfunctional protein.
III. Insertion of Artificial Chromosomes Containing Human Immunoglobulin Genes
• Artificial Chromosomes
• One aspect of the present invention provides ungulates and ungulate cells that lack at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the processes, sequences and/or constructs described herein, which are further modified to express at least part of a human antibody (i.e. immunoglobulin (Ig)) locus. This human locus can undergo rearrangement and express a diverse population of human antibody molecules in the ungulate. These cloned, transgenic ungulates provide a replenishable, theoretically infinite supply of human antibodies (such as polyclonal antibodies), which can be used for therapeutic, diagnostic, purification, and other clinically relevant purposes.
• In one particular embodiment, artificial chromosome (ACs) can be used to accomplish the transfer of human immunoglobulin genes into ungulate cells and animals. ACs permit targeted integration of megabase size DNA fragments that contain single or multiple genes. The ACs, therefore, can introduce heterologous DNA into selected cells for production of the gene product encoded by the heterologous DNA. In a one embodiment, one or more ACs with integrated human immunoglobulin DNA can be used as a vector for introduction of human Ig genes into ungulates (such as pigs).
• First constructed in yeast in 1983, ACs are man-made linear DNA molecules constructed from essential cis-acting DNA sequence elements that are responsible for the proper replication and partitioning of natural chromosomes (Murray et al. (1983), Nature 301:189-193). A chromosome requires at least three elements to function. Specifically, the elements of an artificial chromosome include at least: (1) autonomous replication sequences (ARS) (having properties of replication origins—which are the sites for initiation of DNA replication), (2) centromeres (site of kinetochore assembly that is responsible for proper distribution of replicated chromosomes at mitosis and meiosis), and (3) telomeres (specialized structures at the ends of linear chromosomes that function to both stabilize the ends and facilitate the complete replication of the extreme termini of the DNA molecule).
• In one embodiment, the human Ig can be maintained as an independent unit (an episome) apart from the ungulate chromosomal DNA. For example, episomal vectors contain the necessary DNA sequence elements required for DNA replication and maintenance of the vector within the cell. Episomal vectors are available commercially (see, for example, Maniatis, T. et al., Molecular Cloning, A Laboratory Manual (1982) pp. 368-369). The AC can stably replicate and segregate along side endogenous chromosomes. In an alternative embodiment, the human IgG DNA sequences can be integrated into the ungulate cell's chromosomes thereby permitting the new information to be replicated and partitioned to the cell's progeny as a part of the natural chromosomes (see, for example, Wigler et al. (1977), Cell 11:223). The AC can be translocated to, or inserted into, the endogenous chromosome of the ungulate cell. Two or more ACs can be introduced to the host cell simultaneously or sequentially.
• ACs, furthermore, can provide an extra-genomic locus for targeted integration of megabase size DNA fragments that contain single or multiple genes, including multiple copies of a single gene operatively linked to one promoter or each copy or several copies linked to separate promoters. ACs can permit the targeted integration of megabase size DNA fragments that contain single or multiple human immunoglobulin genes. The ACs can be generated by culturing the cells with dicentric chromosomes (i.e., chromosomes with two centromeres) under such conditions known to one skilled in the art whereby the chromosome breaks to form a minichromosome and formerly dicentric chromosome.
• ACs can be constructed from humans (human artificial chromosomes: “HACs”), yeast (yeast artificial chromosomes: “YACs”), bacteria (bacterial artificial chromosomes: “BACs”), bacteriophage P1-derived artificial chromosomes: “PACs”) and other mammals (mammalian artificial chromosomes: “MACs”). The ACs derive their name (e.g., YAC, BAC, PAC, MAC, HAC) based on the origin of the centromere. A YAC, for example, can derive its centromere from S. cerevisiae. MACs, on the other hand, include an active mammalian centromere while HACs refer to chromosomes that include human centromeres. Furthermore, plant artificial chromosomes (“PLACs”) and insect artificial chromosomes can also be constructed. The ACs can include elements derived from chromosomes that are responsible for both replication and maintenance. ACs, therefore, are capable of stably maintaining large genomic DNA fragments such as human Ig DNA.
• In one embodiment, ungulates containing YACs are provided. YACs are genetically engineered circular chromosomes that contain elements from yeast chromosomes, such as S. cerevisiae, and segments of foreign DNAs that can be much larger than those accepted by conventional cloning vectors (e.g., plasmids, cosmids). YACs allow the propagation of very large segments of exogenous DNA (Schlessinger, D. (1990), Trends in Genetics 6:248-253) into mammalian cells and animals (Choi et al. (1993), Nature Gen 4:117-123). YAC transgenic approaches are very powerful and are greatly enhanced by the ability to efficiently manipulate the cloned DNA. A major technical advantage of yeast is the ease with which specific genome modifications can be made via DNA-mediated transformation and homologous recombination (Ramsay, M. (1994), Mol Biotech 1:181-201). In one embodiment, one or more YACs with integrated human Ig DNA can be used as a vector for introduction of human Ig genes into ungulates (such as pigs).
• The YAC vectors contain specific structural components for replication in yeast, including: a centromere, telomeres, autonomous replication sequence (ARS), yeast selectable markers (e.g., TRP1, URA3, and SUP4), and a cloning site for insertion of large segments of greater than 50 kb of exogenous DNA. The marker genes can allow selection of the cells carrying the YAC and serve as sites for the synthesis of specific restriction endonucleases. For example, the TRP1 and URA3 genes can be used as dual selectable markers to ensure that only complete artificial chromosomes are maintained. Yeast selectable markers can be carried on both sides of the centromere, and two sequences that seed telomere formation in vivo are separated. Only a fraction of one percent of a yeast cell's total DNA is necessary for replication, however, including the center of the chromosome (the centromere, which serves as the site of attachment between sister chromatids and the sites of spindle fiber attachment during mitosis), the ends of the chromosome (telomeres, which serve as necessary sequences to maintain the ends of eukaryotic chromosomes), and another short stretch of DNA called the ARS which serves as DNA segments where the double helix can unwind and begin to copy itself.
• In one embodiment, YACs can be used to clone up to about 1, 2, or 3 Mb of immunoglobulin DNA. In another embodiment, at least 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, or 95 kilobases.
• Yeast integrating plasmids, replicating vectors (which are fragments of YACs), can also be used to express human Ig. The yeast integrating plasmid can contain bacterial plasmid sequences that provide a replication origin and a drug-resistance gene for growth in bacteria (e.g., E. coli), a yeast marker gene for selection of transformants in yeast, and restriction sites for inserting Ig sequences. Host cells can stably acquire this plasmid by integrating it directly into a chromosome. Yeast replicating vectors can also be used to express human Ig as free plasmid circles in yeast. Yeast or ARS-containing vectors can be stabilized by the addition of a centromere sequence. YACs have both centromeric and telomeric regions, and can be used for cloning very large pieces of DNA because the recombinant is maintained essentially as a yeast chromosome.
• YACs are provided, for example, as disclosed in U.S. Pat. Nos. 6,692,954, 6,495,318, 6,391,642, 6,287,853, 6,221,588, 6,166,288, 6,096,878, 6,015,708, 5,981,175, 5,939,255, 5,843,671, 5,783,385, 5,776,745, 5,578,461, and 4,889,806; European Patent Nos. 1 356 062 and 0 648 265; PCT Publication Nos. WO 03/025222, WO 02/057437, WO 02/101044, WO 02/057437, WO 98/36082, WO 98/12335, WO 98/01573, WO 96/01276, WO 95/14769, WO 95/05847, WO 94/23049, and WO 94/00569.
• In another embodiment, ungulates containing BACs are provided. BACs are F-based plasmids found in bacteria, such as E. Coli, that can transfer approximately 300 kb of foreign DNA into a host cell. Once the Ig DNA has been cloned into the host cell, the newly inserted segment can be replicated along with the rest of the plasmid. As a result, billions of copies of the foreign DNA can be made in a very short time. In a particular embodiment, one or more BACs with integrated human Ig DNA are used as a vector for introduction of human Ig genes into ungulates (such as pigs).
• The BAC cloning system is based on the E. coli F-factor, whose replication is strictly controlled and thus ensures stable maintenance of large constructs (Willets, N., and R. Skurray (1987), Structure and function of the F-factor and mechanism of conjugation. In Escherichia coli and Salmonella Typhimurium: Cellular and Molecular Biology (F. C. Neidhardt, Ed) Vol. 2 pp 1110-1133, Am. Soc. Microbiol., Washington, D.C.). BACs have been widely used for cloning of DNA from various eukaryotic species (Cai et al. (1995), Genomics 29:413-425; Kim et al. (1996), Genomics 34:213-218; Misumi et al. (1997), Genomics 40:147-150; Woo et al. (1994), Nucleic Acids Res 22:4922-4931; Zimmer, R. and Gibbins, A. M. V. (1997), Genomics 42:217-226). The low occurrence of the F-plasmid can reduce the potential for recombination between DNA fragments and can avoid the lethal overexpression of cloned bacterial genes. BACs can stably maintain the human immunoglobulin genes in a single copy vector in the host cells, even after 100 or more generations of serial growth.
• BAC (or pBAC) vectors can accommodate inserts in the range of approximately 30 to 300 kb pairs. One specific type of BAC vector, pBeloBac11, uses a complementation of the lacZ gene to distinguish insert-containing recombinant molecules from colonies carrying the BAC vector, by color. When a DNA fragment is cloned into the lacZ gene of pBeloBac11, insertional activation results in a white colony on X-Gal/IPTG plates after transformation (Kim et al. (1996), Genomics 34:213-218) to easily identify positive clones.
• For example, BACs can be provided such as disclosed in U.S. Pat. Nos. 6,713,281, 6,703,198, 6,649,347, 6,638,722, 6,586,184, 6,573,090, 6,548,256, 6,534,262, 6,492,577, 6,492,506, 6,485,912, 6,472,177, 6,455,254, 6,383,756, 6,277,621, 6,183,957, 6,156,574, 6,127,171, 5,874,259, 5,707,811, and 5,597,694; European Patent Nos. 0 805 851; PCT Publication Nos. WO 03/087330, WO 02/00916, WO 01/39797, WO 01/04302, WO 00/79001, WO 99/54487, WO 99/27118, and WO 96/21725.
• In another embodiment, ungulates containing bacteriophage PACs are provided. In a particular embodiment, one or more bacteriophage PACs with integrated human Ig DNA are used as a vector for introduction of human Ig genes into ungulates (such as pigs). For example, PACs can be provided such as disclosed in U.S. Pat. Nos. 6,743,906, 6,730,500, 6,689,606, 6,673,909, 6,642,207, 6,632,934, 6,573,090, 6,544,768, 6,489,458, 6,485,912, 6,469,144, 6,462,176, 6,413,776, 6,399,312, 6,340,595, 6,287,854, 6,284,882, 6,277,621, 6,271,008, 6,187,533, 6,156,574, 6,153,740, 6,143,949, 6,017,755, and 5,973,133; European Patent Nos. 0 814 156; PCT Publication Nos. WO 03/091426, WO 03/076573, WO 03/020898, WO 02/101022, WO 02/070696, WO 02/061073, WO 02/31202, WO 01/44486, WO 01/07478, WO 01/05962, and WO 99/63103.
• In a further embodiment, ungulates containing MACs are provided. MACs possess high mitotic stability, consistent and regulated gene expression, high cloning capacity, and non-immunogenicity. Mammalian chromosomes can be comprised of a continuous linear strand of DNA ranging in size from approximately 50 to 250 Mb. The DNA construct can further contain one or more sequences necessary for the DNA construct to multiply in yeast cells. The DNA construct can also contain a sequence encoding a selectable marker gene. The DNA construct can be capable of being maintained as a chromosome in a transformed cell with the DNA construct. MACs provide extra-genomic specific integration sites for introduction of genes encoding proteins of interest and permit megabase size DNA integration so that, for example, genes encoding an entire metabolic pathway, a very large gene [e.g., such as the cystic fibrosis (CF) gene (˜600 kb)], or several genes [e.g., a series of antigens for preparation of a multivalent vaccine] can be stably introduced into a cell.
• Mammalian artificial chromosomes [MACs] are provided. Also provided are artificial chromosomes for other higher eukaryotic species, such as insects and fish, produced using the MACS are provided herein. Methods for generating and isolating such chromosomes. Methods using the MACs to construct artificial chromosomes from other species, such as insect and fish species are also provided. The artificial chromosomes are fully functional stable chromosomes. Two types of artificial chromosomes are provided. One type, herein referred to as SATACs [satellite artificial chromosomes] are stable heterochromatic chromosomes, and the another type are minichromosomes based on amplification of euchromatin. As used herein, a formerly dicentric chromosome is a chromosome that is produced when a dicentric chromosome fragments and acquires new telomeres so that two chromosomes, each having one of the centromeres, are produced. Each of the fragments can be replicable chromosomes.
• Also provided are artificial chromosomes for other higher eukaryotic species, such as insects and fish, produced using the MACS are provided herein. In one embodiment, SATACs [satellite artificial chromosomes] are provided. SATACs are stable heterochromatic chromosomes. In another embodiment, minichromosomes are provided wherein the minichromosomes are based on amplification of euchromatin.
• In one embodiment, artificial chromosomes can be generated by culturing the cells with the dicentric chromosomes under conditions whereby the chromosome breaks to form a minichromosome and formerly dicentric chromosome. In one embodiment, the SATACs can be generated from the minichromosome fragment, see, for example, in U.S. Pat. No. 5,288,625. In another embodiment, the SATACs can be generated from the fragment of the formerly dicentric chromosome. The SATACs can be made up of repeating units of short satellite DNA and can be fully heterochromatic. In one embodiment, absent insertion of heterologous or foreign DNA, the SATACs do not contain genetic information. In other embodiments, SATACs of various sizes are provided that are formed by repeated culturing under selective conditions and subcloning of cells that contain chromosomes produced from the formerly dicentric chromosomes. These chromosomes can be based on repeating units 7.5 to 10 Mb in size, or megareplicons. These megareplicons can be tandem blocks of satellite DNA flanked by heterologous non-satellite DNA. Amplification can produce a tandem array of identical chromosome segments [each called an amplicon] that contain two inverted megareplicons bordered by heterologous [“foreign”] DNA. Repeated cell fusion, growth on selective medium and/or BrdU [5-bromodeoxyuridine] treatment or other genome destabilizing reagent or agent, such as ionizing radiation, including X-rays, and subcloning can result in cell lines that carry stable heterochromatic or partially heterochromatic chromosomes, including a 150-200 Mb “sausage” chromosome, a 500-1000 Mb gigachromosome, a stable 250-400 Mb megachromosome and various smaller stable chromosomes derived therefrom. These chromosomes are based on these repeating units and can include human immunoglobulin DNA that is expressed. (See also U.S. Pat. No. 6,743,967
• In other embodiments, MACs can be provided, for example, as disclosed in U.S. Pat. Nos. 6,743,967, 6,682,729, 6,569,643, 6,558,902, 6,548,287, 6,410,722, 6,348,353, 6,297,029, 6,265,211, 6,207,648, 6,150,170, 6,150,160, 6,133,503, 6,077,697, 6,025,155, 5,997,881, 5,985,846, 5,981,225, 5,877,159, 5,851,760, and 5,721,118; PCT Publication Nos. WO 04/066945, WO 04/044129, WO 04/035729, WO 04/033668, WO 04/027075, WO 04/016791, WO 04/009788, WO 04/007750, WO 03/083054, WO 03/068910, WO 03/068909, WO 03/064613, WO 03/052050, WO 03/027315, WO 03/023029, WO 03/012126, WO 03/006610, WO 03/000921, WO 02/103032, WO 02/097059, WO 02/096923, WO 02/095003, WO 02/092615, WO 02/081710, WO 02/059330, WO 02/059296, WO 00/18941, WO 97/16533, and WO 96/40965.
• In another aspect of the present invention, ungulates and ungulate cells containing HACs are provided. In a particular embodiment, one or more HACs with integrated human Ig DNA are used as a vector for introduction of human Ig genes into ungulates (such as pigs). In a particular embodiment, one or more HACs with integrated human Ig DNA are used to generate ungulates (for example, pigs) by nuclear transfer which express human Igs in response to immunization and which undergo affinity maturation.
• Various approaches may be used to produce ungulates that express human antibodies (“human Ig”). These approaches include, for example, the insertion of a HAC containing both heavy and light chain Ig genes into an ungulate or the insertion of human B-cells or B-cell precursors into an ungulate during its fetal stage or after it is born (e.g., an immune deficient or immune suppressed ungulate) (see, for example, WO 01/35735, filed Nov. 17, 2000, US 02/08645, filed Mar. 20, 2002). In either case, both human antibody producing cells and ungulate antibody-producing B-cells may be present in the ungulate. In an ungulate containing a HAC, a single B-cell may produce an antibody that contains a combination of ungulate and human heavy and light chain proteins. In still other embodiments, the total size of the HAC is at least to approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Mb.
• For example, HACs can be provided such as disclosed in U.S. Pat. Nos. 6,642,207, 6,590,089, 6,566,066, 6,524,799, 6,500,642, 6,485,910, 6,475,752, 6,458,561, 6,455,026, 6,448,041, 6,410,722, 6,358,523, 6,277,621, 6,265,211, 6,146,827, 6,143,566, 6,077,697, 6,025,155, 6,020,142, and 5,972,649; U.S. Pat. Application No. 2003/0037347; PCT Publication Nos. WO 04/050704, WO 04/044156, WO 04/031385, WO 04/016791, WO 03/101396, WO 03/097812, WO 03/093469, WO 03/091426, WO 03/057923, WO 03/057849, WO 03/027638, WO 03/020898, WO 02/092812, and WO 98/27200.
• Additional examples of ACs into which human immunoglobulin sequences can be inserted for use in the invention include, for example, BACs (e.g., pBeloBAC11 or pBAC108L; see, e.g., Shizuya et al. (1992), Proc Natl Acad Sci USA 89(18):8794-8797; Wang et al. (1997), Biotechniques 23(6):992-994), bacteriophage PACs, YACs (see, e.g., Burke (1990), Genet Anal Tech Appl 7(5):94-99), and MACs (see, e.g., Vos (1997), Nat. Biotechnol. 15(12):1257-1259; Ascenzioni et al. (1997), Cancer Lett 118(2):135-142), such as HACs, see also, U.S. Pat. Nos. 6,743,967, 6,716,608, 6,692,954, 6,670,154, 6,642,207, 6,638,722, 6,573,090, 6,492,506, 6,348,353, 6,287,853, 6,277,621, 6,183,957, 6,156,953, 6,133,503, 6,090,584, 6,077,697, 6,025,155, 6,015,708, 5,981,175, 5,874,259, 5,721,118, and 5,270,201; European Patent Nos. 1 437 400, 1 234 024, 1 356 062, 0 959 134, 1 056 878, 0 986 648, 0 648 265, and 0 338 266; PCT Publication Nos. WO 04/013299, WO 01/07478, WO 00/06715, WO 99/43842, WO 99/27118, WO 98/55637, WO 94/00569, and WO 89/09219. Additional examples include those AC provided in, for example, PCT Publication No. WO 02/076508, WO 03/093469, WO 02/097059; WO 02/096923; US Publication Nos US 2003/0113917 and US 2003/003435; and U.S. Pat. No. 6,025,155.
• In other embodiments of the present invention, ACs transmitted through male gametogenesis in each generation. The AC can be integrating or non-integrating. In one embodiment, the AC can be transmitted through mitosis in substantially all dividing cells. In another embodiment, the AC can provide for position independent expression of a human immunogloulin nucleic acid sequence. In a particular embodiment, the AC can have a transmittal efficiency of at least 10% through each male and female gametogenesis. In one particular embodiment, the AC can be circular. In another particular embodiment, the non-integrating AC can be that deposited with the Belgian Coordinated Collections of Microorganisms—BCCM on Mar. 27, 2000 under accession number LMBP 5473 CB. In additional embodiments, methods for producing an AC are provided wherein a mitotically stable unit containing an exogenous nucleic acid transmitted through male gametogenesis is identified; and an entry site in the mitotically stable unit allows for the integration of human immunoglobulin genes into the unit.
• In other embodiments, ACs are provided that include: a functional centromere, a selectable marker and/or a unique cloning site. Tin other embodiments, the AC can exhibit one or more of the following properties: it can segregate stably as an independent chromosome, immunoglobulin sequences can be inserted in a controlled way and can expressed from the AC, it can be efficiently transmitted through the male and female germline and/or the transgenic animals can bear the chromosome in greater than about 30, 40, 50, 60, 70, 80 or 90% of its cells.
• In particular embodiments, the AC can be isolated from fibroblasts (such as any mammalian or human fibroblast) in which it was mitotically stable. After transfer of the AC into hamster cells, a lox (such as loxP) site and a selectable marker site can be inserted. In other embodiments, the AC can maintain mitotic stability, for example, showing a loss of less than about 5, 2, 1, 0.5 or 0.25 percent per mitosis in the absence of selection. See also, US 2003/0064509 and WO 01/77357.
• Xenogenous Immunoglobulin Genes
• In another aspect of the present invention, transgenic ungulates are provided that expresses a xenogenous immunoglobulin loci or fragment thereof, wherein the immunoglobulin can be expressed from an immunoglobulin locus that is integrated within an endogenous ungulate chromosome. In one embodiment, ungulate cells derived from the transgenic animals are provided. In one embodiment, the xenogenous immunoglobulin locus can be inherited by offspring. In another embodiment, the xenogenous immunoglobulin locus can be inherited through the male germ line by offspring. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
• In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogenous immunoglobulin locus. In one embodiment, the xenogenous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
• In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogenous immunoglobulin locus. In one embodiment, the xenogenous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
• In another embodiment, porcine animals are provided that contain an xenogenous immunoglobulin locus. In one embodiment, the xenogenous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
• Human immunoglobulin genes, such as the Ig heavy chain gene (human chromosome 414), Ig kappa chain gene (human chromosome #2) and/or the Ig lambda chain gene (chromosome #22) can be inserted into Acs, as described above. In a particular embodiment, any portion of the human heavy, kappa and/or lambda Ig genes can be inserted into ACs. In one embodiment, the nucleic acid can be at least 70, 80, 90, 95, or 99% identical to the corresponding region of a naturally-occurring nucleic acid from a human. In other embodiments, more than one class of human antibody is produced by the ungulate. In various embodiments, more than one different human Ig or antibody is produced by the ungulate. In one embodiment, an AC containing both a human Ig heavy chain gene and Ig light chain gene, such as an automatic human artificial chromosome (“AHAC,” a circular recombinant nucleic acid molecule that is converted to a linear human chromosome in vivo by an endogenously expressed restriction endonuclease) can be introduced. In one embodiment, the human heavy chain loci and the light chain loci are on different chromosome arms (i.e., on different side of the centromere). In one embodiments, the heavy chain can include the mu heavy chain, and the light chain can be a lambda or kappa light chain. The Ig genes can be introduced simultaneously or sequentially in one or more than one ACs.
• In particular embodiments, the ungulate or ungulate cell expresses one or more nucleic acids encoding all or part of a human Ig gene which undergoes rearrangement and expresses more than one human Ig molecule, such as a human antibody protein. Thus, the nucleic acid encoding the human Ig chain or antibody is in its unrearranged form (that is, the nucleic acid has not undergone V(D)J recombination). In particular embodiments, all of the nucleic acid segments encoding a V gene segment of an antibody light chain can be separated from all of the nucleic acid segments encoding a J gene segment by one or more nucleotides. In a particular embodiment, all of the nucleic acid segments encoding a V gene segment of an antibody heavy chain can be separated from all of the nucleic acid segments encoding a D gene segment by one or more nucleotides, and/or all of the nucleic acid segments encoding a D gene segment of an antibody heavy chain are separated from all of the nucleic acid segments encoding a J gene segment by one or more nucleotides. Administration of an antigen to a transgenic ungulate containing an unrearranged human Ig gene is followed by the rearrangement of the nucleic acid segments in the human Ig gene locus and the production of human antibodies reactive with the antigen.
• In one embodiment, the AC can express a portion or fragment of a human chromosome that contains an immunoglobulin gene. In one embodiment, the AC can express at least 300 or 1300 kb of the human light chain locus, such as described in Davies et al. 1993 Biotechnology 11: 911-914.
• In another embodiment, the AC can express a portion of human chromosome 22 that contains at least the λ light-chain locus, including V_λ gene segments, J_λ gene segments, and the single C_λ gene. In another embodiment, the AC can express at least one V_λ gene segment, at least one J_λ gene segment, and the C_λ gene. In other embodiment, ACs can contain portions of the lambda locus, such as described in Popov et al. J Exp Med. 1999 May 17; 189(10):1611-20.
• In another embodiment, the AC can express a portion of human chromosome 2 that contains at least the κ light-chain locus, including V_κ gene segments, J_κ gene segments and the single C_κ gene. In another embodiment, the AC can express at least one V_κ gene segment, at least one J_κ gene segment and the C_κ gene. In other embodiments, AC containing portions of the kappa light chain locus can be those describe, for example, in Li et al. 2000 J Immunol 164: 812-824 and Li S Proc Natl Acad Sci USA. 1987 June; 84(12):4229-33. In another embodiment, AC containing approximately 1.3 Mb of human kappa locus are provided, such as described in Zou et al FASEB J. 1996 August; 10(10):1227-32.
• In further embodiments, the AC can express a portion of human chromosome 14 that contains at least the human heavy-chain locus, including V_H, D_H, J_H and C_H gene segments. In another embodiment, the AC can express at least one V_H gene segment, at least one D_H gene segment, at least one J_H gene segment and at least one at least one C_H gene segment. In other embodiments, the AC can express at least 85 kb of the human heavy chain locus, such as described in Choi et al. 1993 Nat Gen 4:117-123 and/or Zou et al. 1996 PNAS 96: 14100-14105.
• In other embodiments, the AC can express portions of both heavy and light chain loci, such as, at least 220, 170, 800 or 1020 kb, for example, as disclosed in Green et al. 1994 Nat Gen 7:13-22; Mendez et al 1995 Genomics 26: 294-307; Mendez et al. 1997 Nat Gen 15: 146-156; Green et al. 1998 J Exp Med 188: 483-495 and/or Fishwild et al. 1996 Nat Biotech 14: 845-851. In another embodiment, the AC can express megabase amounts of human immunoglobulin, such as described in Nicholson J Immunol. 1999 Dec. 15; 163(12):6898-906 and Popov Gene. 1996 Oct. 24; 177(1-2):195-201. In addition, in one particular embodiment, MACs derived from human chromosome #14 (comprising the Ig heavy chain gene), human chromosome #2 comprising the Ig kappa chain gene) and human chromosome #22 (comprising the Ig lambda chain gene) can be introduced simultaneously or successively, such as described in US Patent Publication No. 2004/0068760 to Robl et al. In another embodiments, the total size of the MAC is less than or equal to approximately 10, 9, 8, or 7 megabases.
• In a particular embodiment, human Vh, human Dh, human Jh segments and human mu segments of human immunoglobulins in germline configuration can be inserted into an AC, such as a YAC, such that the Vh, Dh, Jh and mu DNA segments form a repertoire of immunoglobulins containing portions which correspond to the human DNA segments, for example, as described in U.S. Pat. No. 5,545,807 to the Babraham Insttitute. Such ACs, after insertion into ungulate cells and generation of ungulates can produce heavy chain immunoglobulins. In one embodiment, these immunoglobulins can form functional heavy chain-light chain immunoglobulins. In another embodiment, these immunoglobulins can be expressed in an amount allowing for recovery from suitable cells or body fluids of the ungulate. Such immunoglobulins can be inserted into yeast artificial chromosome vectors, such as described by Burke, D T, Carle, G F and Olson, M V (1987) “Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors” Science, 236, 806-812, or by introduction of chromosome fragments (such as described by Richer, J and Lo, C W (1989) “Introduction of human DNA into mouse eggs by injection of dissected human chromosome fragments” Science 245, 175-177).
• Additional information on specific ACs containing human immunoglobulin genes can be found in, for example, recent reviews by Giraldo & Montoliu (2001) Transgenic Research 10: 83-103 and Peterson (2003) Expert Reviews in Molecular Medicine 5: 1-25.
• AC Transfer Methods
• The human immunoglobulin genes can be first inserted into ACs and then the human-immunoglobulin-containing ACs can be inserted into the ungulate cells. Alternatively, the ACs can be transferred to an intermediary mammalian cell, such as a CHO cell, prior to insertion into the ungulate call. In one embodiment, the intermediary mammalian cell can also contain and AC and the first AC can be inserted into the AC of the mammalian cell. In particular, a YAC containing human immunoglobulin genes or fragments thereof in a yeast cell can be transferred to a mammalian cell that harbors an MAC. The YAC can be inserted into the MAC. The MAC can then be transferred to an ungulate cell. The human Ig genes can be inserted into ACs by homologous recombination. The resulting AC containing human Ig genes, can then be introduced into ungulate cells. One or more ungulate cells can be selected by techniques described herein or those known in the art, which contain an AC containing a human Ig.
• Suitable hosts for introduction of the ACs are provided herein, which include but are not limited to any animal or plant, cell or tissue thereof, including, but not limited to: mammals, birds, reptiles, amphibians, insects, fish, arachnids, tobacco, tomato, wheat, monocots, dicots and algae. In one embodiment, the ACs can be condensed (Marschall et al Gene Ther. 1999 September; 6(9):1634-7) by any reagent known in the art, including, but not limited to, spermine, spermidine, polyethylenimine, and/or polylysine prior to introduction into cells. The ACs can be introduced by cell fusion or microcell fusion or subsequent to isolation by any method known to those of skill in this art, including but not limited to: direct DNA transfer, electroporation, nuclear transfer, microcell fusion, cell fusion, spheroplast fusion, lipid-mediated transfer, lipofection, liposomes, microprojectile bombardment, microinjection, calcium phosphate precipitation and/or any other suitable method. Other methods for introducing DNA into cells, include nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells. Polycations, such as polybrene and polyornithine, may also be used. For various techniques for transforming mammalian cells, see e.g., Keown et al. Methods in Enzymology (1990) Vol. 185, pp. 527-537; and Mansour et al. (1988) Nature 336:348-352.
• The ACs can be introduced by direct DNA transformation; microinjection in cells or embryos, protoplast regeneration for plants, electroporation, microprojectile gun and other such methods known to one skilled in the art (see, e.g., Weissbach et al. (1988) Methods for Plant Molecular Biology, Academic Press, N.Y., Section VIII, pp. 421-463; Grierson et al. (1988) Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9; see, also U.S. Pat. Nos. 5,491,075; 5,482,928; and 5,424,409; see, also, e.g., U.S. Pat. No. 5,470,708,).
• In particular embodiments, one or more isolated YACs can be used that harbor human Ig genes. The isolated YACs can be condensed (Marschall et al Gene Ther. 1999 September; 6(9):1634-7) by any reagent known in the art, including, but not limited to spermine, spermidine, polyethylenimine, and/or polylysine. The condensed YACs can then be transferred to porcine cells by any method known in the art (for example, microinjection, electroporation, lipid mediated transfection, etc). Alternatively, the condensed YAC can be transferred to oocytes via sperm-mediated gene transfer or intracytoplasmic sperm injection (ICSI) mediated gene transfer. In one embodiment, spheroplast fusion can be used to transfer YACs that harbor human Ig genes to porcine cells.
• In other embodiments of the invention, the AC containing the human Ig can be inserted into an adult, fetal, or embryonic ungulate cell. Additional examples of ungulate cells include undifferentiated cells, such as embryonic cells (e.g., embryonic stem cells), differentiated or somatic cells, such as epithelial cells, neural cells epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, muscle cells, cells from the female reproductive system, such as a mammary gland, ovarian cumulus, granulosa, or oviductal cell, germ cells, placental cell, or cells derived from any organ, such as the bladder, brain, esophagus, fallopian tube, heart, intestines, gallbladder, kidney, liver, lung, ovaries, pancreas, prostate, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, and uterus or any other cell type described herein.
• Site Specific Recombinase Mediated Transfer
• In particular embodiments of the present invention, the transfer of ACs containing human immunoglobulin genes to porcine cells, such as those described herein or known in the art, can be accomplished via site specific recombinase mediated transfer. In one particular embodiment, the ACs can be transferred into porcine fibroblast cells. In another particular embodiment, the ACs can be YACs.
• In other embodiments of the present invention, the circularized DNA, such as an AC, that contain the site specific recombinase target site can be transferred into a cell line that has a site specific recombinase target site within its genome. In one embodiment, the cell's site specific recombinase target site can be located within an exogenous chromosome. The exogenous chromosome can be an artificial chromosome that does not integrate into the host's endogenous genome. In one embodiment, the AC can be transferred via germ line transmission to offspring. In one particular embodiment, a YAC containing a human immunoglobulin gene or fragment thereof can be circularized via a site specific recombinase and then transferred into a host cell that contains a MAC, wherein the MAC contains a site specific recombinase site. This MAC that now contains human immunoglobulin loci or fragments thereof can then be fused with a porcine cell, such as, but not limited to, a fibroblast. The porcine cell can then be used for nuclear transfer.
• In certain embodiments of the present invention, the ACs that contain human immunoglobulin genes or fragments thereof can be transferred to a mammalian cell, such as a CHO cell, prior to insertion into the ungulate call. In one embodiment, the intermediary mammalian cell can also contain and AC and the first AC can be inserted into the AC of the mammalian cell. In particular, a YAC containing human immunoglobulin genes or fragments thereof in a yeast cell can be transferred to a mammalian cell that harbors a MAC. The YAC can be inserted in the MAC. The MAC can then be transferred to an ungulate cell. In particular embodiments, the YAC harboring the human Ig genes or fragments thereof can contain site specific recombinase target sites. The YAC can first be circularized via application of the appropriate site specific recombinase and then inserted into a mammalian cell that contains its own site specific recombinase target site. Then, the site specific recombinase can be applied to integrate the YAC into the MAC in the intermediary mammalian cell. The site specific recombinase can be applied in cis or trans. In particular, the site specific recombinase can be applied in trans. In one embodiment, the site specific recombinase can be expressed via transfection of a site specific recombinase expression plasmid, such as a Cre expression plasmid. In addition, one telomere region of the YAC can also be retrofitted with a selectable marker, such as a selectable marker described herein or known in the art. The human Ig genes or fragments thereof within the MAC of the intermediary mammalian cell can then be transferred to an ungulate cell, such as a fibroblast.
• Alternatively, the AC, such as a YAC, harboring the human Ig genes or fragments thereof can contain site specific recombinase target sites optionally located near each telomere. The YAC can first be circularized via application of the appropriate site specific recombinase and then inserted into an ungulate cell directly that contains its own site specific recombinase target site within it genome. Alternatively, the ungulate cell can harbor its own MAC, which contains a site specific recombinase target site. In this embodiment, the YAC can be inserted directly into the endogenous genome of the ungulate cell. In particular embodiments, the ungulate cell can be a fibroblast cell or any other suitable cell that can be used for nuclear transfer. See, for example, FIG. 7; Call et al., Hum Mol Genet. 2000 Jul. 22; 9(12):1745-51.
• In other embodiments, methods to circularize at least 100 kb of DNA are provided wherein the DNA can then be integrated into a host genome via a site specific recombinase. In one embodiment, at least 100, 200, 300, 400, 500, 1000, 2000, 5000, 10,000 kb of DNA can be circularized. In another embodiment, at least 1000, 2000, 5000, 10,000, or 20,000 megabases of DNA can be circularized. In one embodiment, the circularization of the DNA can be accomplished by attaching site specific recombinase target sites at each end of the DNA sequence and then applying the site specific recombinase to result in circularization of the DNA. In one embodiment, the site specific recombinase target site can be lox. In another embodiment, the site specific recombinase target site can be Flt. In certain embodiments, the DNA can be an artificial chromosome, such as a YAC or any AC described herein or known in the art. In another embodiment, the AC can contain human immunoglobulin loci or fragments thereof.
• In another preferred embodiment, the YAC can be converted to, or integrated within, an artificial mammalian chromosome. The mammalian artificial chromosome is either transferred to or harbored within a porcine cell. The artificial chromosome can be introduced within the porcine genome through any method known in the art including but not limited to direct injection of metaphase chromosomes, lipid mediated gene transfer, or microcell fusion.
• Site-specific recombinases include enzymes or recombinases that recognize and bind to a short nucleic acid site or sequence-specific recombinase target site, i.e., a recombinase recognition site, and catalyze the recombination of nucleic acid in relation to these sites. These enzymes include recombinases, transposases and integrases. Examples of sequence-specific recombinase target sites include, but are not limited to, lox sites, att sites, dif sites and frt sites. Non-limiting examples of site-specific recombinases include, but are not limited to, bacteriophage P1 Cre recombinase, yeast FLP recombinase, Inti integrase, bacteriophage λ, phi 80, P22, P2, 186, and P4 recombinase, Tn3 resolvase, the Hin recombinase, and the Cin recombinase, E. coli xerC and xerD recombinases, Bacillus thuringiensis recombinase, TpnI and the β-lactamase transposons, and the immunoglobulin recombinases.
• In one embodiment, the recombination site can be a lox site that is recognized by the Cre recombinase of bacteriophage P1. Lox sites refer to a nucleotide sequence at which the product of the cre gene of bacteriophage P1, the Cre recombinase, can catalyze a site-specific recombination event. A variety of lox sites are known in the art, including the naturally occurring loxP, loxB, loxL and loxR, as well as a number of mutant, or variant, lox sites, such as loxP511, loxP514, lox.DELTA.86, lox.DELTA.117, loxC2, loxP2, loxP3 and lox P23. Additional example of lox sites include, but are not limited to, loxB, loxL, loxR, loxP, loxP3, loxP23, loxΔ86, loxΔ117, loxP511, and loxC2.
• In another embodiment, the recombination site is a recombination site that is recognized by a recombinases other than Cre. In one embodiment, the recombinase site can be the FRT sites recognized by FLP recombinase of the 2 pi plasmid of Saccharomyces cerevisiae. FRT sites refer to a nucleotide sequence at which the product of the FLP gene of the yeast 2 micron plasmid, FLP recombinase, can catalyze site-specific recombination. Additional examples of the non-Cre recombinases include, but are not limited to, site-specific recombinases include: att sites recognized by the Int recombinase of bacteriophage λ (e.g. att1, att2, att3, attP, attB, attL, and attR), the recombination sites recognized by the resolvase family, and the recombination site recognized by transposase of Bacillus thuringiensis.
IV. Production of Genetically Modified Animals
• In additional aspects of the present invention, ungulates that contain the genetic modifications described herein can be produced by any method known to one skilled in the art. Such methods include, but are not limited to: nuclear transfer, intracytoplasmic sperm injection, modification of zygotes directly and sperm mediated gene transfer.
• In another embodiment, a method to clone such animals, for example, pigs, includes: enucleating an oocyte, fusing the oocyte with a donor nucleus from a cell in which at least one allele of at least one immunoglobulin gene has been inactivated, and implanting the nuclear transfer-derived embryo into a surrogate mother.
• Alternatively, a method is provided for producing viable animals that lack any expression of functional immunoglobulin by inactivating both alleles of the immunoglobulin gene in embryonic stem cells, which can then be used to produce offspring.
• In another aspect, the present invention provides a method for producing viable animals, such as pigs, in which both alleles of the immunoglobulin gene have been rendered inactive. In one embodiment, the animals are produced by cloning using a donor nucleus from a cell in which both alleles of the immunoglobulin gene have been inactivated. In one embodiment, both alleles of the immunoglobulin gene are inactivated via a genetic targeting event.
• Genetically altered animals that can be created by modifying zygotes directly. For mammals, the modified zygotes can be then introduced into the uterus of a pseudopregnant female capable of carrying the animal to term. For example, if whole animals lacking an immunoglobulin gene are desired, then embryonic stem cells derived from that animal can be targeted and later introduced into blastocysts for growing the modified cells into chimeric animals. For embryonic stem cells, either an embryonic stem cell line or freshly obtained stem cells can be used.
• In a suitable embodiment of the invention, the totipotent cells are embryonic stem (ES) cells. The isolation of ES cells from blastocysts, the establishing of ES cell lines and their subsequent cultivation are carried out by conventional methods as described, for example, by Doetchmann et al., J. Embryol. Exp. Morph. 87:27-45 (1985); Li et al., Cell 69:915-926 (1992); Robertson, E. J. “Tetracarcinomas and Embryonic Stem Cells: A Practical Approach,” ed. E. J. Robertson, IRL Press, Oxford, England (1987); Wurst and Joyner, “Gene Targeting: A Practical Approach,” ed. A. L. Joyner, IRL Press, Oxford, England (1993); Hogen et al., “Manipulating the Mouse Embryo: A Laboratory Manual,” eds. Hogan, Beddington, Costantini and Lacy, Cold Spring Harbor Laboratory Press, New York (1994); and Wang et al., Nature 336:741-744 (1992). In another suitable embodiment of the invention, the totipotent cells are embryonic germ (EG) cells. Embryonic Germ cells are undifferentiated cells functionally equivalent to ES cells, that is they can be cultured and transfected in vitro, then contribute to somatic and germ cell lineages of a chimera (Stewart et al., Dev. Biol. 161:626-628 (1994)). EG cells are derived by culture of primordial germ cells, the progenitors of the gametes, with a combination of growth factors: leukemia inhibitory factor, steel factor and basic fibroblast growth factor (Matsui et al., Cell 70:841-847 (1992); Resnick et al., Nature 359:550-551 (1992)). The cultivation of EG cells can be carried out using methods described in the article by Donovan et al., “Transgenic Animals, Generation and Use,” Ed. L. M. Houdebine, Harwood Academic Publishers (1997), and in the original literature cited therein.
• Tetraploid blastocysts for use in the invention may be obtained by natural zygote production and development, or by known methods by electrofusion of two-cell embryos and subsequently cultured as described, for example, by James et al., Genet. Res. Camb. 60:185-194 (1992); Nagy and Rossant, “Gene Targeting: A Practical Approach,” ed. A. L. Joyner, IRL Press, Oxford, England (1993); or by Kubiak and Tarkowski, Exp. Cell Res. 157:561-566 (1985).
• The introduction of the ES cells or EG cells into the blastocysts can be carried out by any method known in the art. A suitable method for the purposes of the present invention is the microinjection method as described by Wang et al., EMBO J. 10:2437-2450 (1991).
• Alternatively, by modified embryonic stem cells transgenic animals can be produced. The genetically modified embryonic stem cells can be injected into a blastocyst and then brought to term in a female host mammal in accordance with conventional techniques. Heterozygous progeny can then be screened for the presence of the alteration at the site of the target locus, using techniques such as PCR or Southern blotting. After mating with a wild-type host of the same species, the resulting chimeric progeny can then be cross-mated to achieve homozygous hosts.
• After transforming embryonic stem cells with the targeting vector to alter the immunoglobulin gene, the cells can be plated onto a feeder layer in an appropriate medium, e.g., fetal bovine serum enhanced DMEM. Cells containing the construct can be detected by employing a selective medium, and after sufficient time for colonies to grow, colonies can be picked and analyzed for the occurrence of homologous recombination. Polymerase chain reaction can be used, with primers within and without the construct sequence but at the target locus. Those colonies which show homologous recombination can then be used for embryo manipulating and blastocyst injection. Blastocysts can be obtained from superovulated females. The embryonic stem cells can then be trypsinized and the modified cells added to a droplet containing the blastocysts. At least one of the modified embryonic stem cells can be injected into the blastocoel of the blastocyst. After injection, at least one of the blastocysts can be returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct. The blastocysts are selected for different parentage from the transformed ES cells. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected, and then genotyping can be conducted to probe for the presence of the modified immunoglobulin gene.
• In other embodiments, sperm mediated gene transfer can be used to produce the genetically modified ungulates described herein. The methods and compositions described herein to either eliminate expression of endogenous immunoglobulin genes or insert xenogenous immunoglobulin genes can be used to genetically modify the sperm cells via any technique described herein or known in the art. The genetically modified sperm can then be used to impregnate a female recipient via artificial insemination, intracytoplasmic sperm injection or any other known technique. In one embodiment, the sperm and/or sperm head can be incubated with the exogenous nucleic acid for a sufficient time period. Sufficient time periods include, for example, about 30 seconds to about 5 minutes, typically about 45 seconds to about 3 minutes, more typically about 1 minute to about 2 minutes. In particular embodiments, the expression of xenogenous, such as human, immunoglobulin genes in ungulates as described herein, can be accomplished via intracytoplasmic sperm injection.
• The potential use of sperm cells as vectors for gene transfer was first suggested by Brackett et al., Proc., Natl. Acad. Sci. USA 68:353-357 (1971). This was followed by reports of the production of transgenic mice and pigs after in vitro fertilization of oocytes with sperm that had been incubated by naked DNA (see, for example, Lavitrano et al., Cell 57:717-723 (1989) and Gandolfi et al. Journal of Reproduction and Fertility Abstract Series 4, 10 (1989)), although other laboratories were not able to repeat these experiments (see, for example, Brinster et al. Cell 59:239-241 (1989) and Gavora et al., Canadian Journal of Animal Science 71:287-291 (1991)). Since then, there have been several reports of successful sperm mediated gene transfer in chicken (see, for example, Nakanishi and Iritani, Mol. Reprod. Dev. 36:258-261 (1993)); mice (see, for example, Maione, Mol. Reprod. Dev. 59:406 (1998)); and pigs (see, for example, Lavitrano et al. Transplant. Proc. 29:3508-3509 (1997); Lavitrano et al., Proc. Natl. Acad. Sci. USA 99:14230-5 (2002); Lavitrano et al., Mol. Reprod. Dev. 64-284-91 (2003)). Similar techniques are also described in U.S. Pat. No. 6,376,743; issued Apr. 23, 2002; U.S. Patent Publication Nos. 20010044937, published Nov. 22, 2001, and 20020108132, published Aug. 8, 2002.
• In other embodiments, intracytoplasmic sperm injection can be used to produce the genetically modified ungulates described herein. This can be accomplished by co-inserting an exogenous nucleic acid and a sperm into the cytoplasm of an unfertilized oocyte to form a transgenic fertilized oocyte, and allowing the transgenic fertilized oocyte to develop into a transgenic embryo and, if desired, into a live offspring. The sperm can be a membrane-disrupted sperm head or a demembranated sperm head. The co-insertion step can include the substep of preincubating the sperm with the exogenous nucleic acid for a sufficient time period, for example, about 30 seconds to about 5 minutes, typically about 45 seconds to about 3 minutes, more typically about 1 minute to about 2 minutes. The co-insertion of the sperm and exogenous nucleic acid into the oocyte can be via microinjection. The exogenous nucleic acid mixed with the sperm can contain more than one transgene, to produce an embryo that is transgenic for more than one transgene as described herein. The intracytoplasmic sperm injection can be accomplished by any technique known in the art, see, for example, U.S. Pat. No. 6,376,743. In particular embodiments, the expression of xenogenous, such as human, immunoglobulin genes in ungulates as described herein, can be accomplished via intracytoplasmic sperm injection.
• Any additional technique known in the art may be used to introduce the transgene into animals. Such techniques include, but are not limited to pronuclear microinjection (see, for example, Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (see, for example, Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (see, for example, Thompson et al., 1989, Cell 56:313-321; Wheeler, M. B., 1994, WO 94/26884); electroporation of embryos (see, for example, Lo, 1983, Mol Cell. Biol. 3:1803-1814); cell gun; transfection; transduction; retroviral infection; adenoviral infection; adenoviral-associated infection; liposome-mediated gene transfer; naked DNA transfer; and sperm-mediated gene transfer (see, for example, Lavitrano et al., 1989, Cell 57:717-723); etc. For a review of such techniques, see, for example, Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229. In particular embodiments, the expression of xenogenous, such as human, immunoglobulin genes in ungulates as described herein, can be accomplished via these techniques.
• Somatic Cell Nuclear Transfer to Produce Cloned, Transgenic Offspring
• In a further aspect of the present invention, ungulate, such as porcine or bovine, cells lacking one allele, optionally both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be used as donor cells for nuclear transfer into recipient cells to produce cloned, transgenic animals. Alternatively, ungulate heavy chain, kappa light chain and/or lambda light chain gene knockouts can be created in embryonic stem cells, which are then used to produce offspring. Offspring lacking a single allele of a functional ungulate heavy chain, kappa light chain and/or lambda light chain gene produced according to the process, sequences and/or constructs described herein can be breed to further produce offspring lacking functionality in both alleles through mendelian type inheritance.
• In another embodiment, the present invention provides a method for producing viable pigs that lack any expression of functional alpha-1,3-GT by breeding a male pig heterozygous for the alpha-1,3-GT gene with a female pig heterozygous for the alpha-1,3-GT gene. In one embodiment, the pigs are heterozygous due to the genetic modification of one allele of the alpha-1,3-GT gene to prevent expression of that allele. In another embodiment, the pigs are heterozygous due to the presence of a point mutation in one allele of the alpha-1,3-GT gene. In another embodiment, the point mutation can be a T-to-G point mutation at the second base of exon 9 of the alpha-1,3-GT gene. In one specific embodiment, a method to produce a porcine animal that lacks any expression of functional alpha-1,3-GT is provided wherein a male pig that contains a T-to-G point mutation at the second base of exon 9 of the alpha-1,3-GT gene is bred with a female pig that contains a T-to-G point mutation at the second base of exon 9 of the alpha-1,3-GT gene, or vise versa.
• The present invention provides a method for cloning an animal, such as a pig, lacking a functional immunoglobulin gene via somatic cell nuclear transfer. In general, the animal can be produced by a nuclear transfer process comprising the following steps: obtaining desired differentiated cells to be used as a source of donor nuclei; obtaining oocytes from the animal; enucleating said oocytes; transferring the desired differentiated cell or cell nucleus into the enucleated oocyte, e.g., by fusion or injection, to form NT units; activating the resultant NT unit; and transferring said cultured NT unit to a host animal such that the NT unit develops into a fetus.
• Nuclear transfer techniques or nuclear transplantation techniques are known in the art (Dai et al. Nature Biotechnology 20:251-255; Polejaeva et al Nature 407:86-90 (2000); Campbell et al, Theriogenology, 43:181 (1995); Collas et al, Mol. Report Dev., 38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994); Sims et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432, U.S. Pat. Nos. 4,944,384 and 5,057,420).
• A donor cell nucleus, which has been modified to alter the immunoglobulin gene, is transferred to a recipient oocyte. The use of this method is not restricted to a particular donor cell type. The donor cell can be as described herein, see also, for example, Wilmut et al Nature 385 810 (1997); Campbell et al Nature 380 64-66 (1996); Dai et al., Nature Biotechnology 20:251-255, 2002 or Cibelli et al Science 280 1256-1258 (1998). All cells of normal karyotype, including embryonic, fetal and adult somatic cells which can be used successfully in nuclear transfer can be employed. Fetal fibroblasts are a particularly useful class of donor cells. Generally suitable methods of nuclear transfer are described in Campbell et al Theriogenology 43 181 (1995), Dai et al. Nature Biotechnology 20:251-255, Polejaeva et al Nature 407:86-90 (2000), Collas et al Mol. Reprod. Dev. 38 264-267 (1994), Keefer et al Biol. Reprod. 50 935-939 (1994), Sims et al Proc. Nat'l. Acad. Sci. USA 90 6143-6147 (1993), WO-A-9426884, WO-A-9424274, WO-A-9807841, WO-A-9003432, U.S. Pat. No. 4,994,384 and U.S. Pat. No. 5,057,420. Differentiated or at least partially differentiated donor cells can also be used. Donor cells can also be, but do not have to be, in culture and can be quiescent. Nuclear donor cells which are quiescent are cells which can be induced to enter quiescence or exist in a quiescent state in vivo. Prior art methods have also used embryonic cell types in cloning procedures (Campbell et al (Nature, 380:64-68, 1996) and Stice et al (Biol. Reprod., 20 54:100-110, 1996).
• Somatic nuclear donor cells may be obtained from a variety of different organs and tissues such as, but not limited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and a disaggregated preparation of a whole or part of an embryo, fetus, or adult animal. In a suitable embodiment of the invention, nuclear donor cells are selected from the group consisting of epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, extended cells, cumulus cells, epidermal cells or endothelial cells. In another embodiment, the nuclear donor cell is an embryonic stem cell. In a particular embodiment, fibroblast cells can be used as donor cells.
• In another embodiment of the invention, the nuclear donor cells of the invention are germ cells of an animal. Any germ cell of an animal species in the embryonic, fetal, or adult stage may be used as a nuclear donor cell. In a suitable embodiment, the nuclear donor cell is an embryonic germ cell.
• Nuclear donor cells may be arrested in any phase of the cell cycle (G0, G1, G2, S, M) so as to ensure coordination with the acceptor cell. Any method known in the art may be used to manipulate the cell cycle phase. Methods to control the cell cycle phase include, but are not limited to, G0 quiescence induced by contact inhibition of cultured cells, G0 quiescence induced by removal of serum or other essential nutrient, G0 quiescence induced by senescence, G0 quiescence induced by addition of a specific growth factor; G0 or G1 quiescence induced by physical or chemical means such as heat shock, hyperbaric pressure or other treatment with a chemical, hormone, growth factor or other substance; S-phase control via treatment with a chemical agent which interferes with any point of the replication procedure; M-phase control via selection using fluorescence activated cell sorting, mitotic shake off, treatment with microtubule disrupting agents or any chemical which disrupts progression in mitosis (see also Freshney, R. I. “Culture of Animal Cells: A Manual of Basic Technique,” Alan R. Liss, Inc, New York (1983).
• Methods for isolation of oocytes are well known in the art. Essentially, this can comprise isolating oocytes from the ovaries or reproductive tract of an animal. A readily available source of oocytes is slaughterhouse materials. For the combination of techniques such as genetic engineering, nuclear transfer and cloning, oocytes must generally be matured in vitro before these cells can be used as recipient cells for nuclear transfer, and before they can be fertilized by the sperm cell to develop into an embryo. This process generally requires collecting immature (prophase I) oocytes from mammalian ovaries, e.g., bovine ovaries obtained at a slaughterhouse, and maturing the oocytes in a maturation medium prior to fertilization or enucleation until the oocyte attains the metaphase II stage, which in the case of bovine oocytes generally occurs about 18-24 hours post-aspiration. This period of time is known as the “maturation period”. In certain embodiments, the oocyte is obtained from a gilt. A “gilt” is a female pig that has never had offspring. In other embodiments, the oocyte is obtained from a sow. A “sow” is a female pig that has previously produced offspring.
• A metaphase II stage oocyte can be the recipient oocyte, at this stage it is believed that the oocyte can be or is sufficiently “activated” to treat the introduced nucleus as it does a fertilizing sperm. Metaphase II stage oocytes, which have been matured in vivo have been successfully used in nuclear transfer techniques. Essentially, mature metaphase II oocytes can be collected surgically from either non-superovulated or superovulated animal 35 to 48, or 39-41, hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone. The oocyte can be placed in an appropriate medium, such as a hyaluronidase solution.
• After a fixed time maturation period, which ranges from about 10 to 40 hours, about 16-18 hours, about 40-42 hours or about 39-41 hours, the oocytes can be enucleated. Prior to enucleation the oocytes can be removed and placed in appropriate medium, such as HECM containing 1 milligram per milliliter of hyaluronidase prior to removal of cumulus cells. The stripped oocytes can then be screened for polar bodies, and the selected metaphase II oocytes, as determined by the presence of polar bodies, are then used for nuclear transfer. Enucleation follows.
• Enucleation can be performed by known methods, such as described in U.S. Pat. No. 4,994,384. For example, metaphase II oocytes can be placed in either HECM, optionally containing 7.5 micrograms per milliliter cytochalasin B, for immediate enucleation, or can be placed in a suitable medium, for example an embryo culture medium such as CR1aa, plus 10% estrus cow serum, and then enucleated later, such as not more than 24 hours later, or not more than 16-18 hours later.
• Enucleation can be accomplished microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm. The oocytes can then be screened to identify those of which have been successfully enucleated. One way to screen the oocytes is to stain the oocytes with 1 microgram per milliliter 33342 Hoechst dye in HECM, and then view the oocytes under ultraviolet irradiation for less than 10 seconds. The oocytes that have been successfully enucleated can then be placed in a suitable culture medium, for example, CR1aa plus 10% serum.
• A single mammalian cell of the same species as the enucleated oocyte can then be transferred into the perivitelline space of the enucleated oocyte used to produce the NT unit. The mammalian cell and the enucleated oocyte can be used to produce NT units according to methods known in the art. For example, the cells can be fused by electrofusion. Electrofusion is accomplished by providing a pulse of electricity that is sufficient to cause a transient breakdown of the plasma membrane. This breakdown of the plasma membrane is very short because the membrane reforms rapidly. Thus, if two adjacent membranes are induced to breakdown and upon reformation the lipid bilayers intermingle, small channels can open between the two cells. Due to the thermodynamic instability of such a small opening, it enlarges until the two cells become one. See, for example, U.S. Pat. No. 4,997,384 by Prather et al. A variety of electrofusion media can be used including, for example, sucrose, mannitol, sorbitol and phosphate buffered solution. Fusion can also be accomplished using Sendai virus as a fusogenic agent (Graham, Wister Inot. Symp. Monogr., 9, 19, 1969). Also, the nucleus can be injected directly into the oocyte rather than using electroporation fusion. See, for example, Collas and Barnes, Mol. Reprod. Dev., 38:264-267 (1994). After fusion, the resultant fused NT units are then placed in a suitable medium until activation, for example, CR1aa medium. Typically activation can be effected shortly thereafter, for example less than 24 hours later, or about 4-9 hours later, or optimally 1-2 hours after fusion. In a particular embodiment, activation occurs at least one hour post fusion and at 40-41 hours post maturation.
• The NT unit can be activated by known methods. Such methods include, for example, culturing the NT unit at sub-physiological temperature, in essence by applying a cold, or actually cool temperature shock to the NT unit. This can be most conveniently done by culturing the NT unit at room temperature, which is cold relative to the physiological temperature conditions to which embryos are normally exposed. Alternatively, activation can be achieved by application of known activation agents. For example, penetration of oocytes by sperm during fertilization has been shown to activate prefusion oocytes to yield greater numbers of viable pregnancies and multiple genetically identical calves after nuclear transfer. Also, treatments such as electrical and chemical shock can be used to activate NT embryos after fusion. See, for example, U.S. Pat. No. 5,496,720, to Susko-Parrish et al. Fusion and activation can be induced by application of an AC pulse of 5 V for 5 s followed by two DC pulses of 1.5 kV/cm for 60 μs each using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, Calif.). Additionally, activation can be effected by simultaneously or sequentially by increasing levels of divalent cations in the oocyte, and reducing phosphorylation of cellular proteins in the oocyte. This can generally be effected by introducing divalent cations into the oocyte cytoplasm, e.g., magnesium, strontium, barium or calcium, e.g., in the form of an ionophore. Other methods of increasing divalent cation levels include the use of electric shock, treatment with ethanol and treatment with caged chelators. Phosphorylation can be reduced by known methods, for example, by the addition of kinase inhibitors, e.g., serine-threonine kinase inhibitors, such as 6-dimethyl-aminopurine, staurosporine, 2-aminopurine, and sphingosine. Alternatively, phosphorylation of cellular proteins can be inhibited by introduction of a phosphatase into the oocyte, e.g., phosphatase 2A and phosphatase 2B.
• The activated NT units, or “fused embryos”, can then be cultured in a suitable in vitro culture medium until the generation of cell colonies. Culture media suitable for culturing and maturation of embryos are well known in the art. Examples of known media, which can be used for embryo culture and maintenance, include Ham's F-10+10% fetal calf serum (FCS), Tissue Culture Medium-199 (TCM-199)+10% fetal calf serum, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and Whitten's media, and, in one specific example, the activated NT units can be cultured in NCSU-23 medium for about 1-4 h at approximately 38.6° C. in a humidified atmosphere of 5% CO2.
• Afterward, the cultured NT unit or units can be washed and then placed in a suitable media contained in well plates which can contain a suitable confluent feeder layer. Suitable feeder layers include, by way of example, fibroblasts and epithelial cells. The NT units are cultured on the feeder layer until the NT units reach a size suitable for transferring to a recipient female, or for obtaining cells which can be used to produce cell colonies. These NT units can be cultured until at least about 2 to 400 cells, about 4 to 128 cells, or at least about 50 cells.
• Activated NT units can then be transferred (embryo transfers), zero (0)-144 hours post activation, to the oviduct of an female pigs. In one embodiment, the female pigs can be an estrus-synchronized recipient gilt. Crossbred gilts (large white/Duroc/Landrace) (280-400 lbs) can be used. The gilts can be synchronized as recipient animals by oral administration of 18-20 mg Regu-Mate (Altrenogest, Hoechst, Warren, N.J.) mixed into the feed. Regu-Mate can be fed for 14 consecutive days. One thousand units of Human Chorionic Gonadotropin (hCG, Intervet America, Millsboro, Del.) can then be administered i.m. about 105 h after the last Regu-Mate treatment. Embryo transfers can then be performed about 22-26 h after the hCG injection. In one embodiment, the pregnancy can be brought to term and result in the birth of live offspring. In another embodiment, the pregnancy can be terminated early and embryonic cells can be harvested.
• Breeding for Desired Homozygous Knockout Animals
• In another aspect, the present invention provides a method for producing viable animals that lack any expression of a functional immunoglobulin gene is provided by breeding a male heterozygous for the immunoglobulin gene with a female heterozygous for the immunoglobulin gene. In one embodiment, the animals are heterozygous due to the genetic modification of one allele of the immunoglobulin gene to prevent expression of that allele. In another embodiment, the animals are heterozygous due to the presence of a point mutation in one allele of the alpha-immunoglobulin gene. In further embodiments, such heterozygous knockouts can be bred with an ungulate that expresses xenogenous immunoglobulin, such as human. In one embodiment, a animal can be obtained by breeding a transgenic ungulate that lacks expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof with an ungulate that expresses an xenogenous immunoglobulin. In another embodiment, a animal can be obtained by breeding a transgenic ungulate that lacks expression of one allele of heavy chain, kappa light chain and lambda light chain with an ungulate that expresses an xenogenous, such as human, immunoglobulin. In a further embodiment, an animal can be obtained by breeding a transgenic ungulate that lacks expression of one allele of heavy chain, kappa light chain and lambda light chain and expresses an xenogenous, such as human, immunoglobulin with another transgenic ungulate that lacks expression of one allele of heavy chain, kappa light chain and lambda light chain with an ungulate and expresses an xenogenous, such as human, immunoglobulin to produce a homozygous transgenic ungulate that lacks expression of both alleles of heavy chain, kappa light chain and lambda light chain and expresses an xenogenous, such as human, immunoglobulin. Methods to produce such animals are also provided.
• In one embodiment, sexually mature animals produced from nuclear transfer from donor cells that carrying a homozygous knockout in the immunoglobulin gene, can be bred and their offspring tested for the homozygous knockout. These homozygous knockout animals can then be bred to produce more animals.
• In another embodiment, oocytes from a sexually mature homozygous knockout animal can be in vitro fertilized using wild type sperm from two genetically diverse pig lines and the embryos implanted into suitable surrogates. Offspring from these matings can be tested for the presence of the knockout, for example, they can be tested by cDNA sequencing, and/or PCR. Then, at sexual maturity, animals from each of these litters can be mated. In certain methods according to this aspect of the invention, pregnancies can be terminated early so that fetal fibroblasts can be isolated and further characterized phenotypically and/or genotypically. Fibroblasts that lack expression of the immunoglobulin gene can then be used for nuclear transfer according to the methods described herein to produce multiple pregnancies and offspring carrying the desired homozygous knockout.
Additional Genetic Modifications
• In other embodiments, animals or cells lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can contain additional genetic modifications to eliminate the expression of xenoantigens. The additional genetic modifications can be made by further genetically modifying cells obtained from the transgenic cells and animals described herein or by breeding the animals described herein with animals that have been further genetically modified. Such animals can be modified to eliminate the expression of at least one allele of the alpha-1,3-galactosyltransferase gene, the CMP-Neu5Ac hydroxylase gene (see, for example, U.S. Ser. No. 10/863,116), the iGb3 synthase gene (see, for example, U.S. Patent Application 60/517,524), and/or the Forssman synthase gene (see, for example, U.S. Patent Application 60/568,922). In additional embodiments, the animals discloses herein can also contain genetic modifications to express fucosyltransferase, sialyltransferase and/or any member of the family of glucosyltransferases. To achieve these additional genetic modifications, in one embodiment, cells can be modified to contain multiple genetic modifications. In other embodiments, animals can be bred together to achieve multiple genetic modifications. In one specific embodiment, animals, such as pigs, lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can be bred with animals, such as pigs, lacking expression of alpha-1,3-galactosyl transferase (for example, as described in WO 04/028243).
• In another embodiment, the expression of additional genes responsible for xenograft rejection can be eliminated or reduced. Such genes include, but are not limited to the CMP-NEUAc Hydroxylase Gene, the isoGloboside 3 Synthase gene, and the Forssman synthase gene. In addition, genes or cDNA encoding complement related proteins, which are responsible for the suppression of complement mediated lysis can also be expressed in the animals and tissues of the present invention. Such genes include, but are not limited to CD59, DAF, MCP and CD46 (see, for example, WO 99/53042; Chen et al. Xenotransplantation, Volume 6 Issue 3 Page 194—August 1999, which describes pigs that express CD59/DAF transgenes; Costa C et al, Xenotransplantation. 2002 January; 9(1):45-57, which describes transgenic pigs that express human CD59 and H-transferase; Zhao L et al.; Diamond L E et al. Transplantation. 2001 Jan. 15; 71(1):132-42, which describes a human CD46 transgenic pigs.
• Additional modifications can include expression of tissue factor pathway inhibitor (TFPI), heparin, antithrombin, hirudin, TFPI, tick anticoagulant peptide, or a snake venom factor, such as described in WO 98/42850 and U.S. Pat. No. 6,423,316, entitled “Anticoagulant fusion protein anchored to cell membrane”; or compounds, such as antibodies, which down-regulate the expression of a cell adhesion molecule by the cells, such as described in WO 00/31126, entitled “Suppression of xenograft rejection by down regulation of a cell adhesion molecules” and compounds in which co-stimulation by signal 2 is prevented, such as by administration to the organ recipient of a soluble form of CTLA-4 from the xenogeneic donor organism, for example as described in WO 99/57266, entitled “Immunosuppression by blocking T cell co-stimulation signal 2 (B7/CD28 interaction)”.
• In one embodiment, the animals or cells lacking expression of functional immunoglobulin, produced according to the present invention, can be further modified to transgenically express a cytoxic T-lymphocyte associated protein 4-immunoglobin (CTLA4). The animals or cells can be modified to express CTLA4 peptide or a biologically active fragment (e.g., extracellular domain, truncated form of the peptide in which at least the transmembrane domain has been removed) or derivative thereof. The peptide may be, e.g., human or porcine. The CTLA4 peptide can be mutated. Mutated peptides may have higher affinity than wildtype for porcine and/or human B7 molecules. In one specific embodiment, the mutated CTLA4 can be CTLA4 (Glu104, Tyr29). The CTLA4 peptide can be modified such that it is expressed intracellularly. Other modifications of the CTLA4 peptide include addition of a golgi retention signal to the N or C terminus. The golgi retention signal may be, e.g., the sequence KDEL. The CTLA4 peptide can be fused to a peptide dimerization domain or an immunoglobulin (Ig) molecule. The CTLA4 fusion peptides can include a linker sequence that can join the two peptides.
• Certain aspects of the invention are described in greater detail in the non-limiting Examples that follow.
EXAMPLES Example 1: Porcine Heavy Chain Targeting and Generation of Porcine Animals that Lack Expression of Heavy Chain
• A portion of the porcine Ig heavy-chain locus was isolated from a 3× redundant porcine BAC library. In general, BAC libraries can be generated by fragmenting pig total genomic DNA, which can then be used to derive a BAC library representing at least three times the genome of the whole animal. BACs that contain porcine heavy chain immunoglobulin can then be selected through hybridization of probes selective for porcine heavy chain immunoglobulin as described herein.
• Sequence from a clone (Seq ID 1) was used to generate a primer complementary to a portion of the J-region (the primer is represented by Seq ID No. 2). Separately, a primer was designed that was complementary to a portion of Ig heavy-chain mu constant region (the primer is represented by Seq ID No. 3). These primers were used to amplify a fragment of porcine Ig heavy-chain (represented by Seq ID No. 4) that led the functional joining region (J-region) and sufficient flanking region to design and build a targeting vector. To maintain this fragment and subclones of this fragment in a native state, the E. coli (Stable 2, Invitrogen cat #1026-019) that harbored these fragments was maintained at 30° C. Regions of Seq. ID No. 4 were subcloned and used to assemble a targeting vector as shown in Seq. ID No. 5. This vector was transfected into porcine fetal fibroblasts that were subsequently subjected to selection with G418. Resulting colonies were screened by PCR to detect potential targeting events (Seq ID No. 6 and Seq ID No. 7, 5′ screen primers; and Seq ID No. 8 and Seq ID No. 9, 3′ screen primers). See FIG. 1 for a schematic illustrating the targeting. Targeting was confirmed by southern blotting. Piglets were generated by nuclear transfer using the targeted fetal fibroblasts as nuclear donors.
• Nuclear Transfer.
• The targeted fetal fibroblasts were used as nuclear donor cells. Nuclear transfer was performed by methods that are well known in the art (see, e.g., Dai et al., Nature Biotechnology 20: 251-255, 2002; and Polejaeva et al., Nature 407:86-90, 2000).
• Enucleation of in vitro-matured oocytes (BoMed, Madison, Wis.; TransOva Genetics, Sioux City, Iowa) was begun between 40 and 42 hours post-maturation as described in Polejaeva, I. A., et al. (Nature 407, 86-90 (2000)). For enucleation, we incubated the oocytes in calcium-free phosphate-buffered NCSU-23 medium containing 5 μg ml⁻¹ cytochalasin B (Sigma) and 7.5 μg ml⁻¹ Hoechst 33342 (Sigma) at 38° C. for 20 min. A small amount of cytoplasm from directly beneath the first polar body was then aspirated using an 18 μM glass pipette (Humagen, Charlottesville, Va.). We exposed the aspirated karyoplast to ultraviolet light to confirm the presence of a metaphase plate.
• For nuclear transfer, a single fibroblast cell was placed under the zona pellucida in contact with each enucleated oocyte. Fusion and activation were induced by application of an AC pulse of 5 V for 5 s followed by two DC pulses of 1.5 kV/cm for 60 μs each using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, Calif.). Fused embryos were cultured in NCSU-23 medium for 1-4 h at 38.6° C. in a humidified atmosphere of 5% CO₂, and then transferred to the oviduct of an estrus-synchronized recipient gilt. Crossbred gilts (large white/Duroc/landrace) (280-400 lbs) were synchronized as recipients by oral administration of 18-20 mg Regu-Mate (Altrenogest, Hoechst, Warren, N.J.) mixed into their feed. Regu-Mate was fed for 14 consecutive days. Human chorionic gonadotropin (hCG, 1,000 units; Intervet America, Millsboro, Del.) was administered intra-muscularly 105 h after the last Regu-Mate treatment. Embryo transfers were done 22-26 h after the hCG injection.
• Nuclear transfer produced 18 healthy piglets from four litters. These animals have one functional wild-type Ig heavy-chain locus and one disrupted Ig heavy chain locus.

• Seq ID 2: primer from	ggccagacttcctcggaacagctca
  Butler subclone to
  amplify J to C
  heavychain (637Xba5′)
  Seq ID 3: primer for C	ttccaggagaaggtgacggagct
  to amplify J to C
  heavychain (JM1L)
  Seq ID 6: heavychain 5′	tctagaagacgctggagagaggccag
  primer for 5′ screen
  (HCKOXba5′2)
  Seq ID 7: heavychain 3′	taaagcgcatgctccagactgcctt
  primer for 5′ screen
  (5′arm5′)
  Seq ID 8: heavychain 5′	catcgccttctatcgccttctt
  primer for 3′ screen
  (NEO4425)
  Seq ID 9: heavychain 3′	Aagtacttgccgcctctcagga
  primer for 3′ screen
  (650 + CA)

• Southern Blot Analysis of Cell and Pig Tissue Samples.
• Cells or tissue samples were lysed overnight at 60° C. in lysis buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% (w/v) Sarcosyl, 1 mg/ml proteinase K) and the DNA precipitated with ethanol. The DNA was then digested with NcoI or XbaI, depending on the probe to be used, and separated on a 1% agarose gel. After electrophoresis, the DNA was transferred to a nylon membrane and probed with digoxigenin-labeled probe (SEQ ID No 41 for NcoI digest, SEQ ID No 40 for XbaI digest). Bands were detected using a chemiluminescent substrate system (Roche Molecular Biochemicals).
Probes for Heavy Chain Southern:

• HC J Probe (used with Xba I digest)

  (Seq ID No 40)

  CTCTGCACTCACTACCGCCGGACGCGCACTGCCGTGCTGCCCATGGACCA

  CGCTGGGGAGGGGTGAGCGGACAGCACGTTAGGAAGTGTGTGTGTGCGCG

  TGGGTGCAAGTCGAGCCAAGGCCAAGATCCAGGGGCTGGGCCCTGTGCCC

  AGAGGAGAATGGCAGGTGGAGTGTAGCTGGATTGAAAGGTGGCCTGAAGG

  GTGGGGCATCCTGTTTGGAGGCTCACTCTCAGCCCCAGGGTCTCTGGTTC

  CTGCCGGGGTGGGGGGCGCAAGGTGCCTACCACACCCTGCTAGCCCCTCG

  TCCAGTCCCGGGCCTGCCTCTTCACCACGGAAGAGGATAAGCCAGGCTGC

  AGGCTTCATGTGCGCCGTGGAGAACCCAGTTCGGCCCTTGGAGG

  HC Mu Probe (used with NcoI digest)

  (Seq ID No 41)

  GGCTGAAGTCTGAGGCCTGGCAGATGAGCTTGGACGTGCGCTGGGGAGTA

  CTGGAGAAGGACTCCCGGGTGGGGACGAAGATGTTCAAGACGGGGGGCTG

  CTCCTCTACGACTGCAGGCAGGAACGGGGCGTCACTGTGCCGGCGGCACC

  CGGCCCCGCCCCCGCCACAGCCACAGGGGGAGCCCAGCTCACCTGGCCCA

  GAGATGGACACGGACTTGGTGCCACTGGGGTGCTGGACCTCGCACACCAG

  GAAGGCCTCTGGGTCCTGGGGGATGCTCACAGAGGGTAGGAGCACCCGGG

  AGGAGGCCAAGTACTTGCCGCCTCTCAGGACGG

Example 2: Porcine Kappa Light Chain Targeting and Generation of Porcine Lacking Expression of Kappa Light Chain
• A portion of the porcine Ig kappa-chain locus was isolated from a 3× redundant porcine BAC library. In general, BAC libraries can be generated by fragmenting pig total genomic DNA, which can then be used to derive a BAC library representing at least three times the genome of the whole animal. BACs that contain porcine kappa chain immunoglobulin can then be selected through hybridization of probes selective for porcine kappa chain immunoglobulin as described herein.
• A fragment of porcine Ig light-chain kappa was amplified using a primer complementary to a portion of the J-region (the primer is represented by Seq ID No. 10) and a primer complementary to a region of kappa C-region (represented by Seq ID No. 11). The resulting amplimer was cloned into a plasmid vector and maintained in Stable2 cells at 30° C. (Seq ID No. 12). See FIG. 2 for a schematic illustration.
• Separately, a fragment of porcine Ig light-chain kappa was amplified using a primer complementary to a portion of the C-region (Seq ID No. 13) and a primer complementary to a region of the kappa enhancer region (Seq ID No. 14). The resulting amplimer was fragmented by restriction enzymes and DNA fragments that were produced were cloned, maintained in Stable2 cells at 30 degrees C. and sequenced. As a result of this sequencing, two non-overlapping contigs were assembled (Seq ID No. 15, 5′ portion of amplimer; and Seq ID No. 16, 3′ portion of amplimer). Sequence from the downstream contig (Seq ID No. 16) was used to design a set of primers (Seq ID No. 17 and Seq ID No. 18) that were used to amplify a contiguous fragment near the enhancer (Seq ID No. 19). A subclone of each Seq ID No. 12 and Seq ID No. 19 were used to build a targeting vector (Seq ID No. 20). This vector was transfected into porcine fetal fibroblasts that were subsequently subjected to selection with G418. Resulting colonies were screened by PCR to detect potential targeting events (Seq ID No. 21 and Seq ID No. 22, 5′ screen primers; and Seq ID No. 23 and Seq Id No 43, 3′ screen primers, and Seq ID No. 24 and Seq Id No 24, endogenous screen primers). Targeting was confirmed by southern blotting. Southern blot strategy design was facilitated by cloning additional kappa sequence, it corresponds to the template for germline kappa transcript (Seq ID No. 25). Fetal pigs were generated by nuclear transfer.
• Nuclear Transfer.
• The targeted fetal fibroblasts were used as nuclear donor cells. Nuclear transfer was performed by methods that are well known in the art (see, e.g., Dai et al., Nature Biotechnology 20: 251-255, 2002; and Polejaeva et al., Nature 407:86-90, 2000).
• Oocytes were collected 46-54 h after the hCG injection by reverse flush of the oviducts using pre-warmed Dulbecco's phosphate buffered saline (PBS) containing bovine serum albumin (BSA; 4 gl⁻¹) (as described in Polejaeva, I. A., et al. (Nature 407, 86-90 (2000)). Enucleation of in vitro-matured oocytes (BoMed, Madison, Wis.) was begun between 40 and 42 hours post-maturation as described in Polejaeva, I. A., et al. (Nature 407, 86-90 (2000)). Recovered oocytes were washed in PBS containing 4 gl⁻¹ BSA at 38° C., and transferred to calcium-free phosphate-buffered NCSU-23 medium at 38° C. for transport to the laboratory. For enucleation, we incubated the oocytes in calcium-free phosphate-buffered NCSU-23 medium containing 5 μg ml⁻¹ cytochalasin B (Sigma) and 7.5 μg ml⁻¹ Hoechst 33342 (Sigma) at 38° C. for 20 min. A small amount of cytoplasm from directly beneath the first polar body was then aspirated using an 18 μM glass pipette (Humagen, Charlottesville, Va.). We exposed the aspirated karyoplast to ultraviolet light to confirm the presence of a metaphase plate.
• For nuclear transfer, a single fibroblast cell was placed under the zona pellucida in contact with each enucleated oocyte. Fusion and activation were induced by application of an AC pulse of 5 V for 5 s followed by two DC pulses of 1.5 kV/cm for 60 μs each using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, Calif.). Fused embryos were cultured in NCSU-23 medium for 1-4 h at 38.6° C. in a humidified atmosphere of 5% CO₂, and then transferred to the oviduct of an estrus-synchronized recipient gilt. Crossbred gills (large white/Duroc/landrace) (280-400 lbs) were synchronized as recipients by oral administration of 18-20 mg Regu-Mate (Altrenogest, Hoechst, Warren, N.J.) mixed into their feed. Regu-Mate was fed for 14 consecutive days. Human chorionic gonadotropin (hCG, 1,000 units; Intervet America, Millsboro, Del.) was administered intra-muscularly 105 h after the last Regu-Mate treatment. Embryo transfers were done 22-26 h after the hCG injection.
• Nuclear transfer using kappa targeted cells produced 33 healthy pigs from 5 litters. These pigs have one functional wild-type allele of porcine Ig light-chain kappa and one disrupted Ig light-chain kappa allele.

• Seq ID 10: kappa J to C	caaggaqaccaagctggaactc
  5′ primer (kjc5′1)
  Seq ID 11: kappa J to C	tgatcaagcacaccacagagacag
  3′ primer (kjc3′2)
  Seq ID 13: 5′ primer for	gatgccaagccatccgtcttcatc
  Kappa C to E (porKCS1)
  Seq ID 14: 3′ primer for	tgaccaaagcagtgtgacggttgc
  Kappa C to E (porKCA1)
  Seq ID 17: kappa 5′	ggatcaaacacgcatcctcatggac
  primer for amplification
  of enhancer region
  (K3′arm1S)
  Seq ID 18: kappa 3′	ggtgattggggcatggttgagg
  primer for amplification
  of enhancer region
  (K3′arm1A)
  Seq ID 21: kappa screen,	cgaacccctgtgtatatagtt
  5′ primer, 5′
  (kappa5armS)
  Seq ID 22: kappa screen,	gagatgaggaagaggagaaca
  3′ primer, 5′
  (kappaNeoA)
  Seq ID 23: kappa screen,	gcattgtctgagtaggtgtcatt
  5′ primer, 3′
  (kappaNeoS)
  Seq ID 24: kappa screen,	cgcttcttgcagggaacacgat
  3′ primer, 5′
  (kappa5armProbe3′)
  Seq ID No 43, Kappa	GTCTTTGGTTTTTGCTGAGGGTT
  screen, 3′ primer
  (kappa3armA2)

Southern Blot Analysis of Cell and Pig Tissue Samples.
• Cells or tissue samples were lysed overnight at 60° C. in lysis buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% (w/v) Sarcosyl, 1 mg/ml proteinase K) and the DNA precipitated with ethanol. The DNA was then digested with SacI and separated on a 1% agarose gel. After electrophoresis, the DNA was transferred to a nylon membrane and probed with digoxigenin-labeled probe (SEQ ID No 42). Bands were detected using a chemiluminescent substrate system (Roche Molecular Biochemicals).
Probe for Kappa Southern:

• Kappa5ArmProbe 5′/3′

  (SEQ ID No 42)

  gaagtgaagccagccagttcctcctgggcaggtggccaaaattacagttg

  acccctcctggtctggctgaaccttgccccatatggtgacagccatctgg

  ccagggcccaggtctccctctgaagcctttgggaggagagggagagtggc

  tggcccgatcacagatgcggaaggggctgactcctcaaccggggtgcaga

  ctctgcagggtgggtctgggcccaacacacccaaagcacgcccaggaagg

  aaaggcagcttggtatcactgcccagagctaggagaggcaccgggaaaat

  gatctgtccaagacccgttcttgcttctaaactccgagggggtcagatga

  agtggttttgtttcttggcctgaagcatcgtgttccctgcaagaagcgg

Example 3 Characterization of the Porcine Lambda Gene Locus
• To disrupt or disable porcine lambda, a targeting strategy has been devised that allows for the removal or disruption of the region of the lambda locus that includes a concatamer of J to C expression cassettes. BAC clones that contain portions of the porcine genome can be generated. A portion of the porcine Ig lambda-chain locus was isolated from a 3× redundant porcine BAC library. In general, BAC libraries can be generated by fragmenting pig total genomic DNA, which can then be used to derive a BAC library representing at least three times the genome of the whole animal. BACs that contain porcine lambda chain immunoglobulin can then be selected through hybridization of probes selective for porcine lambda chain immunoglobulin as described herein.
• BAC clones containing a lambda J-C flanking region (see FIG. 3), can be independently fragmented and subcloned into a plasmid vector. Individual subclones have been screened by PCR for the presence of a portion of the J to C intron. We have cloned several of these cassettes by amplifying from one C region to the next C region. This amplification was accomplished by using primers that are oriented to allow divergent extension within any one C region (Seq ID 26 and Seq ID 27). To obtain successful amplification, the extended products converge with extended products originated from adjacent C regions (as opposed to the same C region). This strategy produces primarily amplimers that extend from one C to the adjacent C. However, some amplimers are the result of amplification across the adjacent C and into the next C which lies beyond the adjacent C. These multi-gene amplimers contain a portion of a C, both the J and C region of the next J-C unit, the J region of the third J-C unit, and a portion of the C region of the third J-C unit. Seq ID 28 is one such amplimer and represents sequence that must be removed or disrupted.
• Other porcine lambda sequences that have been cloned include: Seq ID No. 32, which includes 5′ flanking sequence to the first lambda J/C unit of the porcine lambda light chain genomic sequence; Seq ID No. 33, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, from approximately 200 base pairs downstream of lambda J/C; Seq ID No. 34, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, approximately 11.8 Kb downstream of the J/C cluster region, near the enhancer; Seq ID No. 35, which includes approximately 12 Kb downstream of lambda, including the enhancer region; Seq ID No. 36, which includes approximately 17.6 Kb downstream of lambda; Seq ID No. 37, which includes approximately 19.1 Kb downstream of lambda; Seq ID No. 38, which includes approximately 21.3 Kb downstream of lambda; and Seq ID No. 39, which includes approximately 27 Kb downstream of lambda.

• Seq ID 26: 5′primer for	ccttcctcctgcacctgtcaac
  lambda C to C amplimer
  (lamC5′)
  Seq ID 27: 3′ primer for	tagacacaccagggtggccttg
  lambda C to C amplimer
  (lamC3′)

Example 4 Production of Targeting Vectors for the Lambda Gene
• Following a first targeting strategy, shown in FIG. 4, a vector is designed and built with one targeting arm that is homologous to a region upstream of J1 (i.e., the first J/C unit or sequence) and the other arm homologous to a region that is downstream of the last C (i.e., the last J/C unit or sequence) This targeting vector utilizes a selectable marker (SM).
• Seq ID No. 48 represents one example of a vector used in the first targeting strategy. Seq ID No. 48 is a lambda light chain knockout vector which includes both 5′ and 3′ homology arms and Neo resistance factor.

• Seq ID No. 48

  GCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATA

  GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG

  TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAG

  CTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGT

  CCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGT

  AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCA

  CGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTC

  TTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACT

  GGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT

  GAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCT

  GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA

  TCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCA

  GCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT

  CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTG

  GTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAA

  ATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACA

  GTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT

  CGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACG

  GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCAC

  GCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCC

  GAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAA

  TTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA

  ACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGT

  ATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATC

  CCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTG

  TCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG

  CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG

  TGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTT

  GCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACT

  TTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAG

  GATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCA

  ACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAA

  ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATG

  TTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGG

  GTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAA

  CAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCAA

  ACAGCTATGACCATGGCGGCCGCgtcgacAGGGTGTGGCCAAATACAGCA

  TGGAGTAGCCATCATAAGGAATCTTACACAAGCCTCCAAAATTGTGTTTC

  TGAAATTGGGTTTAAAGTACGTTTGCATTTTAAAAAGCCTGCCAGAAAAT

  ACAGAAAAATGTCTGTGATATGTCTCTGGCTGATAGGATTTTGCTTAGTT

  TTAATTTTGGCTTTATAATTTTCTATAGTTATGAAAATGTTCACAAGAAG

  ATATATTTCATTTTAGCTTCTAAAATAATTATAACACAGAAGTAATTTGT

  GCTTTAAAAAAATATTCAACACAGAAGTATATAAAGTAAAAATTGAGGAG

  TTCCCATCGTGGCTCAGTGATTAACAAACCCAACTAGTATCCATGAGGAT

  ATGGATTTGATCCCTGGCCTTGCTCAGTGGGTTGAGGATCCAGTGTTGCT

  GTGAGCTGTGGTGTAGGTTGCAGACACAGCACTCTGGCGTTGCTGTGACT

  CTGGCGTAGGCCGGCAGCTACAGCTCCATTTGGACCCTTAGCCTGGGAAC

  CTCCATATGCCTGAGATACGGCCCTAAAAAGTCAAAAGCCAAAAAAATAG

  TAAAAATTGAGTGTTTCTACTTACCACCCCTGCCCACATCTTATGCTAAA

  ACCCGTTCTCCAGAGACAAACATCGTCAGGTGGGTCTATATATTTCCAGC

  CCTCCTCCTGTGTGTGTATGTCCGTAAAACACACACACACACACACACAC

  GCACACACACACACACGTATCTAATTAGCATTGGTATTAGTTTTTCAAAA

  GGGAGGTCATGCTCTACCTTTTAGGCGGCAAATAGATTATTTAAACAAAT

  CTGTTGACATTTTCTATATCAACCCATAAGATCTCCCATGTTCTTGGAAA

  GGCTTTGTAAGACATCAACATCTGGGTAAACCAGCATGGTTTTTAGGGGG

  TTGTGTGGATTTTTTTCATATTTTTTAGGGCACACCTGCAGCATATGGAG

  GTTCCCAGGCTAGGGGTTGAATCAGAGCTGTAGCTGCCGGCCTACACCAC

  AGCCACAGCAACGCCAGATCCTTAACCCACTGAGAAAGGCCAGGGATTGA

  ACCTGCATCCTCATGGATGCTGGTCAGATTTATTTCTGCTGAGCCACAAC

  AGGAACTCCCTGAACCAGAATGCTTTTAACCATTCCACTTTGCATGGACA

  TTTAGATTGTTTCCATTTAAAAATACAAATTACAAGGAGTTCCCGTCGTG

  GCTCAGTGGTAACGAATTGGACTAGGAACCATGAGGTTTCGGGTTCGATC

  CCTGGCCTTGCTCGGTGGGTTAAGGATCCAGCATTGATGTGAGATATGGT

  GTAGGTCGCAGACGTGGCTCGGATCCCACGTTGCTGTGGCTCTGGCGTAG

  GCCGGCAACAACAGCTCCGATTCGACCCCTAGCCTGGGAACCTCCATGTG

  CCACAGGAGCAGCCCTAGAAAAGGCAAAAAGACAAAAAAATAAAAAATTA

  AAATGAAAAAATAAAATAAAAATACAAATTACAAGAGACGGCTACAAGGA

  AATCCCCAAGTGTGTGCAAATGCCATATATGTATAAAATGTACTAGTGTC

  TCCTCGCGGGAAAGTTGCCTAAAAGTGGGTTGGCTGGACAGAGAGGACAG

  GCTTTGACATTCTCATAGGTAGTAGCAATGGGCTTCTCAAAATGCTGTTC

  CAGTTTACACTCACCATAGCAAATGACAGTGCCTCTTCCTCTCCACCCTT

  GCCAATAATGTGACAGGTGGATCTTTTTCTATTTTGTGTATCTGACAAGC

  AAAAAATGAGAACAGGAGTTCCTGTCGTGGTGCAGTGGAGACAAATCTGA

  CTAGGAACCATGAAATTTCGGGTTCAATCCCTGGCCTCACTCAGTAGGTA

  AAGGATCCAGGGTTGCAGTGAGCTGTGGGGTAGGTCGCAGACACAGTGCA

  AATTTGGCCCTGTTGTGGCTGTGGTGTAGGCCGGCAGCTATAGCTCCAAT

  TGGACCCCTAGCCTGGGAACCTCCTTATGCCGTGGGTGAGGCCCTAAAAA

  AAAGAGTGCAAAAAAAAAAAATAAGAACAAAAATGATCATCGTTTAATTC

  TTTATTTGATCATTGGTGAAACTTATTTTCCTTTTATATTTTTATTGACT

  GATTTTATTTCTCCTATGAATTTACCGGTCATAGTTTTGCCTGGGTGTTT

  TTACTCCGGTTTTAGTTTTGGTTGGTTGTATTTTCTTAGAGAGCTATAGA

  AACTCTTCATCTATTTGGAATAGTAATTCCTCATTAAGTATTTGTGCTGC

  AAAAAATTTTCCCTGATCTGTTTTATGCTTTTGTTTGTGGGGTCTTTCAC

  GAGAAAGCCTTTTTAGTTTTTACACCTCAGCTTGGTTGTTTTTCTTGATT

  GTGTCTGTAATCTGCGGCCAACATAGGAAACACATTTTTACTTTAGTGTT

  TTTTTCCTATTTTCTTCAAGTACGTCCATTGTTTTGGTGTCTGATTTTAC

  TTTGCCTGGGGTTTGTTTTTGTGTGGCAGGAATATAAACTTATGTATTTT

  CCAAATGGAGAGCCAATGGTTGTATATTTGTTGAATTCAAATGCAACTTT

  ATCAAACACCAAATCATCGATTTATCACAACTCTTCTCTGGTTTATTGAT

  CTAATGATCAATTCCTGTTCCACGCTGTTTTAATTATTTTAGCTTTGTGG

  ATTTTGGTGCCTGGTAGAGAACAAAGCCTCCATTATTTTCATTCAAAATA

  GTCCCGTCTATTATCTGCCATTGTTGTAGTATTAGACTTTAAAATCAATT

  TACTGATTTTCAAAAGTTATTCCTTTGGTGATGTGGAATACTTTATACTT

  CATAAGGTACATGGATTCATTTGTGGGGAATTGATGTCTTTGCTATTGTG

  GCCATTTGTCAAGTTGTGTAATATTTTACCCATGCCAACTTTGCATATTG

  TATGTGAGTTTATTCCCAGGGTTTTTAATAGGATGTTTATTGAAGTTGTC

  AGTGTTTCCACAATTTCATCGCCTCAGTGCTTACTGTTTGCATAAAAGGA

  AACCTACTCACTTTTGCCTATTGCTCTTGTATTCAATCATTTTAGTTAAC

  TCTTGTGTTAATTTTGAGAGTTTTTCAGCTGACTGTCTGGGGTTTTCTTT

  AATAGACTAGCCCTTTGTCTGTAAAGAATAATTTTATCGAATTTTTCTTA

  ACACTCACACTCTCCCCACCCCCACCCCCGCTCATCTCCTTTCATTGGGT

  CAAATCTGTAGAATACAATAAAAGTAAGAGTGGGAACCTTAGCCTTTAAG

  TCGATTTTGCCTTTAAATGTGAATGTTGCTATGTTTCGGGACATTCTCTT

  TATCAAGTTGCGGATGTTTCCTTAGATAATTAACTTAATAAAAGACTGGA

  TGTTTGCTTTCTTCAAATCAGAATTGTGTTGAATTTATATTGCTATTCTG

  TTTAATTTTGTTTCAAAAAATTTACATGCACACCTTAAAGATAACCATGA

  CCAAATAGTCCTCCTGCTGAGAGAAAATGTTGGCCCCAATGCCACAGGTT

  ACCTCCCGACTCAGATAAACTACAATGGGAGATAAAATCAGATTTGGCAA

  AGCCTGTGGATTCTTGCCATAACTCTCAGAGCATGACTTGGGTGTTTTTT

  CCTTTTCTAAGTATTTTAATGGTATTTTTGTGTTACAATAGGAAATCTAG

  GACACAGAGAGTGATTCAATGAGGGGAACGCATTCTGGGATGACTCTAGG

  CCTCTGGTTTGGGGAGAGCTCTATTGAAGTAAAGACAATGAGAGGAAGCA

  AGTTTGCAGGGAACTGTGAGGAATTTAGATGGGGAATGTTGGGTTTGAGG

  TTTCTATAGGGCACGCAAGCAGAGATGCACTCAGGAGGAAGAAGGAGCAT

  AAATCTAGAGGCAAAAAGAGAGGTCAGGACTGGAAATAGAGATGCGAGAC

  ACCAGGGTGGCAGTCAGAGAGCACAGTGTGGGTCAGAAGACAGTGGAAGA

  ACACAAGGGACAGAGAGGGATCTCCAACTTCACTGGGATGAGGGCCTTGT

  TGGCCTTGACCTGAGAGATTTCCAGGAGTTGAGGGTGGGAAGGAGccgcg

  gTCTAGGAAGCTTTCTAGGGTACCTCTAGGGATCCGAACAATGGAAGTCC

  GAGCTCATCGCTAATAACTTCGTATAGCATACATTATACGAAGTTATATT

  CGATGCGGCCGCAAGGGGTTCGCGTCAGCGGGTGTTGGCGGGTGTCGGGG

  CTGGCTTAACTATGCGGCATCAGAGCAGagatccCGGCGCGCCCTACCGG

  GTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGC

  CCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATT

  CCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTC

  GCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCA

  GCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTC

  TCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGG

  GGCAGCGGCCAATAGCAGCTTTGGCTCCTTCGCTTTCTGGGCTCAGAGGC

  TGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGG

  GCGGGCGCCCGAAGGTCCTCCGGAAGCCCGGCATTCTGCACGCTTCAAAA

  GCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGA

  CCTGCAGCCAATATGGGATCGGCCATTGAACAAGATGGATTGCACGCAGG

  TTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAAC

  AGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGG

  CGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACT

  GCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTT

  GCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTA

  TTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGC

  CGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTG

  ATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGA

  GCACGTACTCGGATGGAAGCCGGTCTTGTCAATCAGGATGATCTGGACGA

  AGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGC

  GCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTG

  CCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGG

  CCGGCTGGGTGTGGCGGATCGCTATCAGGACATAGCGTTGGCTACCCGTG

  ATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTT

  TACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCT

  TGACGAGTTCTTCTGAGGGGATCAATTCtctagtGAACAATGGAAGTCCG

  AGCTCATCGCTAATAACTTCGTATAGCATACATTATACGAAGTTATATTC

  GATGCGGCCGCAAGGGGTTCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGC

  TGGCTTAACTATGCGGCATCAGAGCAGtctagaGCTCGCTGATCAGCCTC

  GACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGC

  CTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAAT

  GAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGG

  TGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGC

  ATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGC

  TGGGGGCGCGCCCctcgagGGGAAGGTATCTCCCAGGAAACTGGCCAGGA

  CACATTGGTCCTCCGCCCTCCCCTTCCTCCCACTCCTCCTCCAGACAGGA

  CTGTGCCCACCCCCTGCCACCTTTCTGGCCAGAACTGTCCATGGCAGGTG

  ACCTTCACATGAGCCCTTCCTCCCTGCCTGCCCTAGTGGGACCCTCCATA

  CCTCCCCCTGGACCCCGTTGTCCTTTCTTTCCAGTGTGGCCCTGAGCATA

  ACTGATGCCATCATGGGCTGCTGACCCACCCGGGACTGTGTTGTGCAGTG

  AGTCACTTCTCTGTCATCAGGGCTTTGTAATTGATAGATAGTGTTTCATC

  ATCATTAGGACCGGGTGGCCTCTATGCTCTGTTAGTCTCCAAACACTGAT

  GAAAACCTTCGTTGGCATAGTCCCAGCTTCCTGTTGCCCATCCATAAATC

  TTGACTTAGGGATGCACATCCTGTCTCCAAGCAACCACCCCTCCCCTAGG

  CTAACTATAAAACTGTCCCAATGGCCCTTGTGTGGTGCAGAGTTCATGCT

  TCCAGATCATTTCTCTGCTAGATCCATATCTCACCTTGTAAGTCATCCTA

  TAATAAACTGATCCATTGATTATTTGCTTCTGTTTTTTCCATCTCAAAAC

  AGCTTCTCAGTTCAGTTCGAATTTTTTATTCCCTCCATCCACCCATACTT

  TCCTCAGCCTGGGGAACCCTTGCCCCCAGTCCCATGCCCTTCCTCCCTCT

  CTGCCCAGCTCAGCACCTGCCCACCCTCACCCTTCCTGTCACTCCCTAGG

  ACTGGACCATCCACTGGGGCCAGGACACTCCAGCAGCCTTGGCTTCATGG

  GCTCTGAAATCCATGGCCCATCTCTATTCCTCACTGGATGGCAGGTTCAG

  AGATGTGAAAGGTCTAGGAGGAAGCCAGGAAGGAAACTGTTGCATGAAAG

  GCCGGCCTGATGGTTCAGTACTTAAATAATATGAGCTCTGAGCTCCCCAG

  GAACCAAAGCATGGAGGGAGTATGTGCCTCAGAATCTCTCTGAGATTCAG

  CAAAGCCTTTGCTAGAGGGAAAATAGTGGCTCAACCTTGAGGGCCAGCAT

  CTTGCACCACAGTTAAAAGTGGGTATTTGTTTTACCTGAGGCCTCAGCAT

  TATGGGAACCGGGCTCTGACACAAACACAGGTGCAGCCCGGCAGCCTCAG

  AACACAGCAACGACCACAAGCTGGGACAGCTGCCCCTGAACGGGGAGTCC

  ACCATGCTTCTGTCTCGGGTACCACCAGGTCACCATCCCTGGGGGAGGTA

  GTTCCATAGCAGTAGTCCCCTGATTTCGCCCCTCGGGCGTGTAGCCAGGC

  AAGCTCCTGCCTCTGGACCCAGGGTGGACCCTTGCTCCCCACTACCCTGC

  ACATGCCAGACAGTCAAGACCACTCCCACCTCTGTCTGAGGCCCCCTTGG

  GTGTCCCAGGGCCCCCGAGCTGTCCTCTACTCATGGTTCTTCCACCTGGG

  TACAAAAGAGGCGAGGGACACTTTTCTCAGGTTTGCGGCTCAGAAAGGTA

  CCTTCCTAGGGTTTGTCCACTGGGAGTCACCTCCCTTGCATCTCAATGTC

  AGTGGGGAAAACTGGGTCCCATGGGGGGATTAGTGCCACTGTGAGGCCCC

  TGAAGTCTGGGGCCTCTAGACACTATGATGATGAGGGATGTGGTGAAAAA

  CCCCACCCCAGCCCTTCTTGCCGGGACCCTGGGCTGTGGCTCCCCCATTG

  CACTTGGGGTCAGAGGGGTGGATGGTGGCTATGGTCAGGCATGTTTCCCA

  TGAGCTGGGGGCACCCTGGGTGACTTTCTCCTGTGAATCCTGAATTAGCA

  GCTATAACAAATTGCCCAAACTCTTAGGCTTAAAACAACACACATTTATT

  CCTCTGGGTCCCAGGGTCAGAAGTCCAAAATGAGTCCTATAGGCTAAATT

  TGAGGTGTCTCTGGGTTGAGCTCCTCCTGGAAGCCTTTTCCAGCCTCTAG

  AGTCCCAAGTCCTTGGCTCTGGGCCCCTCCCTCAAGCTTCAAAGCCACAG

  AAGCTTCTAATCTCTCTCCCTTCCCCTCTGACCTCTGCTCCCATCCTCAT

  ACCCTGTCCCCTCACTCTGACCCTCCTGCCTCCCTCTTTCCCTTATAAAG

  ACCCTGCATGGGGCCACGGAGATAATCCAGGGTAATCGCCCCTCTTCCAG

  CCCTTAACTCCATCCCATCTGCAAAATCCCTGTCACCCCATAATGGACCT

  ACagatctCCTAGAGTTAACACTGGCCGTCGTTTTACCGGTCCGTAGTCA

  GGTTTAGTTCGTCCGGCGGCGCCAGAAATCCGCGCGGTGGTTTTTGGGGG

  TCGGGGGTGTTTGGCAGCCACAGACGCCCGGTGTTCGTGTCGCGCCAGTA

  CATGCGGTCCATGCCCAGGCCATCCAAAAACCATGGGTCTGTCTGCTCAG

  TCCAGTCGTGGACTGACCCCACGCAACGCCCAAAATAATAACCCCCACGA

  ACCATAAACCATTCCCCATGGGGGACCCCGTCCCTAACCCACGGGGCCCG

  TGGCTATGGCAGGCCTGCCGCCCGACGTTGGCTGCGAGCCCTGGGCCTTC

  ACCCGAACTTGGGGGGTGGGGTGGGGAAAAGGAAGAAACGCGGGCGTATT

  GGCCCCAATGGGGTCTCGGTGGGGTATCGACAGAGTGCCAGCCCTGGGAC

  CGAACCCCGCGTTTATGAACAAACGACCCAACACCCGTGCGTTTTATTCT

  GTCTTTTTATTGCCGACATAGCGCGGGTTCCTTCCGGTATTGTCTCCTTC

  CGTGTTTCAGTTAGCCTCCCCCATCTCCCGTGCAAACGTGCGCGCCAGGT

  CGCAGATCGTCGGTATGGAGCCTGGGGTGGTGACGTGGGTCTGGATCATC

  CCGGAGGTAAGTTGCAGCAGGGCGTCCCGGCAGCCGGCGGGCGATTGGTC

  GTAATCCAGGATAAAGACGTGCATGGGACGGAGGCGTTTGGCCAAGACGT

  CCAAGGCCCAGGCAAACACGTTGTACAGGTCGCCGTTGGGGGCCAGCAAC

  TCGGGGGCCCGAAACAGGGTAAATAACGTGTCCCCGATATGGGGTCGTGG

  GCCCGCGTTGCTCTGGGGCTCGGCACCCTGGGGCGGCACGGCCGTCCCCG

  AAAGCTGTCCCCAATCCTCCCGCCACGACCCGCCGCCCTGCAGATACCGC

  ACCGTATTGGCAAGCAGCCCGTAAACGCGGCGAATCGCGGTCAGCATAGC

  CAGGTCAAGCCGCTCGCCGGGGCGCTGGCGTTTGGCCAGGCGGTCGATGT

  GTCTGTCCTCCGGAAGGGCCCCCAACACGATGTTTGTGCCGGGCAAGGTC

  GGCGGGATGAGGGCCACGAACGCCAGCACGGCCTGGGGGGTCATGCTGCC

  CATAAGGTATCGCGCGGCCGGGTAGCACAGGAGGGCGGCGATGGGATGGC

  GGTCGAAGATGAGGGTGAGGGCCGGGGGCGGGGCATGTGAGCTCCCAGCC

  TCCCCCCCGATATGAGGAGCCAGAACGGCGTCGGTCACGGTATAAGGCAT

  GCCCATTGTTATCTGGGCGCTTGTCATTACCACCGCCGCGTCCCCGGCCG

  ATATCTCACCCTGGTCAAGGCGGTGTTGTGTGGTGTAGATGTTCGCGATT

  GTCTCGGAAGCCCCCAGCACCCGCCAGTAAGTCATCGGCTCGGGTACGTA

  GACGATATCGTCGCGCGAACCCAGGGCCACCAGCAGTTGCGTGGTGGTGG

  TTTTCCCCATCCCGTGGGGACCGTCTATATAAACCCGCAGTAGCGTGGGC

  ATTTTCTGCTCCGGGCGGACTTCCGTGGCTTCTTGCTGCCGGCGAGGGCG

  CAACGCCGTACGTCGGTTGCTATGGCCGCGAGAACGCGCAGCCTGGTCGA

  ACGCAGACGCGTGCTGATGGCCGGGGTACGAAGCCATACGCGCTTCTACA

  AGGCGCTGGCCGAAGAGGTGCGGGAGTTTCACGCCACCAAGATGTGCGGC

  ACGCTGTTGACGCTGTTAAGCGGGTCGCTGCAGGGTCGCTCGGTGTTCGA

  GGCCACACGCGTCACCTTAATATGCGAAGTGGACCTGGGACCGCGCCGCC

  CCGACTGCATCTGCGTGTTCCAATTCGCCAATGACAAGACGCTGGGCGGG

  GTTTGCTCGACATTGGGTGGAAACATTCCAGGCCTGGGTGGAGAGGCTTT

  TTGCTTCCTCTTGCAAAACCACACTGCTCGACATTGGGTGGAAACATTCC

  AGGCCTGGGTGGAGAGGCTTTTTGCTTCCTCTTGAAAACCACACTGCTCG

  ACTCTACGGTCCG

  
  Seq ID No. 49 is a lambda light chain 5′ arm sequence

• Seq ID No. 49

  AGGGTGTGGCCAAATACAGCATGGAGTAGCCATCATAAGGAATCTTACAC

  AAGCCTCCAAAATTGTGTTTCTGAAATTGGGTTTAAAGTACGTTTGCATT

  TTAAAAAGCCTGCCAGAAAATACAGAAAAATGTCTGTGATATGTCTCTGG

  CTGATAGGATTTTGCTTAGTTTTAATTTTGGCTTTATAATTTTCTATAGT

  TATGAAAATGTTCACAAGAAGATATATTTCATTTTAGCTTCTAAAATAAT

  TATAACACAGAAGTAATTTGTGCTTTAAAAAAATATTCAACACAGAAGTA

  TATAAAGTAAAAATTGAGGAGTTCCCATCGTGGCTCAGTGATTAACAAAC

  CCAACTAGTATCCATGAGGATATGGATTTGATCCCTGGCCTTGCTCAGTG

  GGTTGAGGATCCAGTGTTGCTGTGAGCTGTGGTGTAGGTTGCAGACACAG

  CACTCTGGCGTTGCTGTGACTCTGGCGTAGGCCGGCAGCTACAGCTCCAT

  TTGGACCCTTAGCCTGGGAACCTCCATATGCCTGAGATACGGCCCTAAAA

  AGTCAAAAGCCAAAAAAATAGTAAAAATTGAGTGTTTCTACTTACCACCC

  CTGCCCACATCTTATGCTAAAACCCGTTCTCCAGAGACAAACATCGTCAG

  GTGGGTCTATATATTTCCAGCCCTCCTCCTGTGTGTGTATGTCCGTAAAA

  CACACACACACACACACACACGCACACACACACACACGTATCTAATTAGC

  ATTGGTATTAGTTTTTCAAAAGGGAGGTCATGCTCTACCTTTTAGGCGGC

  AAATAGATTATTTAAACAAATCTGTTGACATTTTCTATATCAACCCATAA

  GATCTCCCATGTTCTTGGAAAGGCTTTGTAAGACATCAACATCTGGGTAA

  ACCAGCATGGTTTTTAGGGGGTTGTGTGGATTTTTTTCATATTTTTTAGG

  GCACACCTGCAGCATATGGAGGTTCCCAGGCTAGGGGTTGAATCAGAGCT

  GTAGCTGCCGGCCTACACCACAGCCACAGCAACGCCAGATCCTTAACCCA

  CTGAGAAAGGCCAGGGATTGAACCTGCATCCTCATGGATGCTGGTCAGAT

  TTATTTCTGCTGAGCCACAACAGGAACTCCCTGAACCAGAATGCTTTTAA

  CCATTCCACTTTGCATGGACATTTAGATTGTTTCCATTTAAAAATACAAA

  TTACAAGGAGTTCCCGTCGTGGCTCAGTGGTAACGAATTGGACTAGGAAC

  CATGAGGTTTCGGGTTCGATCCCTGGCCTTGCTCGGTGGGTTAAGGATCC

  AGCATTGATGTGAGATATGGTGTAGGTCGCAGACGTGGCTCGGATCCCAC

  GTTGCTGTGGCTCTGGCGTAGGCCGGCAACAACAGCTCCGATTCGACCCC

  TAGCCTGGGAACCTCCATGTGCCACAGGAGCAGCCCTAGAAAAGGCAAAA

  AGACAAAAAAATAAAAAATTAAAATGAAAAAATAAAATAAAAATACAAAT

  TACAAGAGACGGCTACAAGGAAATCCCCAAGTGTGTGCAAATGCCATATA

  TGTATAAAATGTACTAGTGTCTCCTCGCGGGAAAGTTGCCTAAAAGTGGG

  TTGGCTGGACAGAGAGGACAGGCTTTGACATTCTCATAGGTAGTAGCAAT

  GGGCTTCTCAAAATGCTGTTCCAGTTTACACTCACCATAGCAAATGACAG

  TGCCTCTTCCTCTCCACCCTTGCCAATAATGTGACAGGTGGATCTTTTTC

  TATTTTGTGTATCTGACAAGCAAAAAATGAGAACAGGAGTTCCTGTCGTG

  GTGCAGTGGAGACAAATCTGACTAGGAACCATGAAATTTCGGGTTCAATC

  CCTGGCCTCACTCAGTAGGTAAAGGATCCAGGGTTGCAGTGAGCTGTGGG

  GTAGGTCGCAGACACAGTGCAAATTTGGCCCTGTTGTGGCTGTGGTGTAG

  GCCGGCAGCTATAGCTCCAATTGGACCCCTAGCCTGGGAACCTCCTTATG

  CCGTGGGTGAGGCCCTAAAAAAAAGAGTGCAAAAAAAAAAAATAAGAACA

  AAAATGATCATCGTTTAATTCTTTATTTGATCATTGGTGAAACTTATTTT

  CCTTTTATATTTTTATTGACTGATTTTATTTCTCCTATGAATTTACCGGT

  CATAGTTTTGCCTGGGTGTTTTTACTCCGGTTTTAGTTTTGGTTGGTTGT

  ATTTTCTTAGAGAGCTATAGAAACTCTTCATCTATTTGGAATAGTAATTC

  CTCATTAAGTATTTGTGCTGCAAAAAATTTTCCCTGATCTGTTTTATGCT

  TTTGTTTGTGGGGTCTTTCACGAGAAAGCCTTTTTAGTTTTTACACCTCA

  GCTTGGTTGTTTTTCTTGATTGTGTCTGTAATCTGCGGCCAACATAGGAA

  ACACATTTTTACTTTAGTGTTTTTTTCCTATTTTCTTCAAGTACGTCCAT

  TGTTTTGGTGTCTGATTTTACTTTGCCTGGGGTTTGTTTTTGTGTGGCAG

  GAATATAAACTTATGTATTTTCCAAATGGAGAGCCAATGGTTGTATATTT

  GTTGAATTCAAATGCAACTTTATCAAACACCAAATCATCGATTTATCACA

  ACTCTTCTCTGGTTTATTGATCTAATGATCAATTCCTGTTCCACGCTGTT

  TTAATTATTTTAGCTTTGTGGATTTTGGTGCCTGGTAGAGAACAAAGCCT

  CCATTATTTTCATTCAAAATAGTCCCGTCTATTATCTGCCATTGTTGTAG

  TATTAGACTTTAAAATCAATTTACTGATTTTCAAAAGTTATTCCTTTGGT

  GATGTGGAATACTTTATACTTCATAAGGTACATGGATTCATTTGTGGGGA

  ATTGATGTCTTTGCTATTGTGGCCATTTGTCAAGTTGTGTAATATTTTAC

  CCATGCCAACTTTGCATATTGTATGTGAGTTTATTCCCAGGGTTTTTAAT

  AGGATGTTTATTGAAGTTGTCAGTGTTTCCACAATTTCATCGCCTCAGTG

  CTTACTGTTTGCATAAAAGGAAACCTACTCACTTTTGCCTATTGCTCTTG

  TATTCAATCATTTTAGTTAACTCTTGTGTTAATTTTGAGAGTTTTTCAGC

  TGACTGTCTGGGGTTTTCTTTAATAGACTAGCCCTTTGTCTGTAAAGAAT

  AATTTTATCGAATTTTTCTTAACACTCACACTCTCCCCACCCCCACCCCC

  GCTCATCTCCTTTCATTGGGTCAAATCTGTAGAATACAATAAAAGTAAGA

  GTGGGAACCTTAGCCTTTAAGTCGATTTTGCCTTTAAATGTGAATGTTGC

  TATGTTTCGGGACATTCTCTTTATCAAGTTGCGGATGTTTCCTTAGATAA

  TTAACTTAATAAAAGACTGGATGTTTGCTTTCTTCAAATCAGAATTGTGT

  TGAATTTATATTGCTATTCTGTTTAATTTTGTTTCAAAAAATTTACATGC

  ACACCTTAAAGATAACCATGACCAAATAGTCCTCCTGCTGAGAGAAAATG

  TTGGCCCCAATGCCACAGGTTACCTCCCGACTCAGATAAACTACAATGGG

  AGATAAAATCAGATTTGGCAAAGCCTGTGGATTCTTGCCATAACTCTCAG

  AGCATGACTTGGGTGTTTTTTCCTTTTCTAAGTATTTTAATGGTATTTTT

  GTGTTACAATAGGAAATCTAGGACACAGAGAGTGATTCAATGAGGGGAAC

  GCATTCTGGGATGACTCTAGGCCTCTGGTTTGGGGAGAGCTCTATTGAAG

  TAAAGACAATGAGAGGAAGCAAGTTTGCAGGGAACTGTGAGGAATTTAGA

  TGGGGAATGTTGGGTTTGAGGTTTCTATAGGGCACGCAAGCAGAGATGCA

  CTCAGGAGGAAGAAGGAGCATAAATCTAGAGGCAAAAAGAGAGGTCAGGA

  CTGGAAATAGAGATGCGAGACACCAGGGTGGCAGTCAGAGAGCACAGTGT

  GGGTCAGAAGACAGTGGAAGAACACAAGGGACAGAGAGGGATCTCCAACT

  TCACTGGGATGAGGGCCTTGTTGGCCTTGACCTGAGAGATTTCCAGGAGT

  TGAGGGTGGGAAGGAG

  
  Seq. ID No. 50 is a lambda 3′ arm sequence

• Seq. ID No. 50

  GGGAAGGTATCTCCCAGGAAACTGGCCAGGACACATTGGTCCTCCGCCCT

  CCCCTTCCTCCCACTCCTCCTCCAGACAGGACTGTGCCCACCCCCTGCCA

  CCTTTCTGGCCAGAACTGTCCATGGCAGGTGACCTTCACATGAGCCCTTC

  CTCCCTGCCTGCCCTAGTGGGACCCTCCATACCTCCCCCTGGACCCCGTT

  GTCCTTTCTTTCCAGTGTGGCCCTGAGCATAACTGATGCCATCATGGGCT

  GCTGACCCACCCGGGACTGTGTTGTGCAGTGAGTCACTTCTCTGTCATCA

  GGGCTTTGTAATTGATAGATAGTGTTTCATCATCATTAGGACCGGGTGGC

  CTCTATGCTCTGTTAGTCTCCAAACACTGATGAAAACCTTCGTTGGCATA

  GTCCCAGCTTCCTGTTGCCCATCCATAAATCTTGACTTAGGGATGCACAT

  CCTGTCTCCAAGCAACCACCCCTCCCCTAGGCTAACTATAAAACTGTCCC

  AATGGCCCTTGTGTGGTGCAGAGTTCATGCTTCCAGATCATTTCTCTGCT

  AGATCCATATCTCACCTTGTAAGTCATCCTATAATAAACTGATCCATTGA

  TTATTTGCTTCTGTTTTTTCCATCTCAAAACAGCTTCTCAGTTCAGTTCG

  AATTTTTTATTCCCTCCATCCACCCATACTTTCCTCAGCCTGGGGAACCC

  TTGCCCCCAGTCCCATGCCCTTCCTCCCTCTCTGCCCAGCTCAGCACCTG

  CCCACCCTCACCCTTCCTGTCACTCCCTAGGACTGGACCATCCACTGGGG

  CCAGGACACTCCAGCAGCCTTGGCTTCATGGGCTCTGAAATCCATGGCCC

  ATCTCTATTCCTCACTGGATGGCAGGTTCAGAGATGTGAAAGGTCTAGGA

  GGAAGCCAGGAAGGAAACTGTTGCATGAAAGGCCGGCCTGATGGTTCAGT

  ACTTAAATAATATGAGCTCTGAGCTCCCCAGGAACCAAAGCATGGAGGGA

  GTATGTGCCTCAGAATCTCTCTGAGATTCAGCAAAGCCTTTGCTAGAGGG

  AAAATAGTGGCTCAACCTTGAGGGCCAGCATCTTGCACCACAGTTAAAAG

  TGGGTATTTGTTTTACCTGAGGCCTCAGCATTATGGGAACCGGGCTCTGA

  CACAAACACAGGTGCAGCCCGGCAGCCTCAGAACACAGCAACGACCACAA

  GCTGGGACAGCTGCCCCTGAACGGGGAGTCCACCATGCTTCTGTCTCGGG

  TACCACCAGGTCACCATCCCTGGGGGAGGTAGTTCCATAGCAGTAGTCCC

  CTGATTTCGCCCCTCGGGCGTGTAGCCAGGCAAGCTCCTGCCTCTGGACC

  CAGGGTGGACCCTTGCTCCCCACTACCCTGCACATGCCAGACAGTCAAGA

  CCACTCCCACCTCTGTCTGAGGCCCCCTTGGGTGTCCCAGGGCCCCCGAG

  CTGTCCTCTACTCATGGTTCTTCCACCTGGGTACAAAAGAGGCGAGGGAC

  ACTTTTCTCAGGTTTGCGGCTCAGAAAGGTACCTTCCTAGGGTTTGTCCA

  CTGGGAGTCACCTCCCTTGCATCTCAATGTCAGTGGGGAAAACTGGGTCC

  CATGGGGGGATTAGTGCCACTGTGAGGCCCCTGAAGTCTGGGGCCTCTAG

  ACACTATGATGATGAGGGATGTGGTGAAAAACCCCACCCCAGCCCTTCTT

  GCCGGGACCCTGGGCTGTGGCTCCCCCATTGCACTTGGGGTCAGAGGGGT

  GGATGGTGGCTATGGTCAGGCATGTTTCCCATGAGCTGGGGGCACCCTGG

  GTGACTTTCTCCTGTGAATCCTGAATTAGCAGCTATAACAAATTGCCCAA

  ACTCTTAGGCTTAAAACAACACACATTTATTCCTCTGGGTCCCAGGGTCA

  GAAGTCCAAAATGAGTCCTATAGGCTAAATTTGAGGTGTCTCTGGGTTGA

  GCTCCTCCTGGAAGCCTTTTCCAGCCTCTAGAGTCCCAAGTCCTTGGCTC

  TGGGCCCCTCCCTCAAGCTTCAAAGCCACAGAAGCTTCTAATCTCTCTCC

  CTTCCCCTCTGACCTCTGCTCCCATCCTCATACCCTGTCCCCTCACTCTG

  ACCCTCCTGCCTCCCTCTTTCCCTTATAAAGACCCTGCATGGGGCCACGG

  AGATAATCCAGGGTAATCGCCCCTCTTCCAGCCCTTAACTCCATCCCATC

  TGCAAAATCCCTGTCACCCCATAATGGACCTAC

• In a second strategy, the targeting strategy utilizes a vector pair. One targeting vector is designed to target upstream of J1. See FIG. 5. This targeting vector utilizes a selectable marker that can be selected for or against. Any combination of positive and negative selectable markers described herein or known in the art can be used. A fusion gene composed of the coding region of Herpes simplex thymidine kinase (TK) and the Tn5 aminoglycoside phosphotransferase (Neo resistance) genes is used. This fusion gene is flanked by recognition sites for any site specific recombinase (SSRRS) described herein or known in the art, such as lox sites. Upon isolation of targeted cells through the use of G418 selection, Cre is supplied in trans to delete the marker gene (See FIG. 5). Cells that have deleted the marker gene are selected by addition of any drug known in the art that can be metabolized by TK into a toxic product, such as ganciclovir. The resulting genotype is then targeted with a second vector. The second targeting vector (FIG. 6) is designed to target downstream of last C and uses a positive/negative selection system that is flanked on only one side by a specific recombination site (lox). The recombination site is placed distally in relation to the first targeting event. Upon isolation of the targeted genotype, Cre is again supplied in trans to mediate deletion from recombination site provided in the first targeting event to the recombination site delivered in the second targeting event. The entire J to C cluster region will be removed. The appropriate genotype is again selected by administration of ganciclovir.
• Two vector pairs, i.e., lambda targeting constructs, were designed and built to target the first and last J/C regions and to include site-specific recombination sites. The first vector pair was composed of Seq ID No. 44 (step 1 vector) and Seq ID No. 45 (step 2 vector). The second vector pair was composed of Seq ID No. 46 (step 2 vector) and Seq ID No. 47 (step 1 vector).
Overview of Seq ID No. 44 (Upstream Vector, Step 1, Double Lox): Feature Map
• CDS (3 total)
• • NEO (+STOP) CDS
    • Start: 3311 End: 4114 (Complementary)
  • TK CDS (from VEC1198)
    • Start: 4118 End: 5251 (Complementary)
  • AP(R)
    • Start: 11732 End: 12589 (Complementary)
    • bla gene—Ap(r) determinant
• Enhancer (1 total)
• • CMV Enhancer
    • Start: 5779 End: 6199 (Complementary)
• Misc. Binding Site (2 total)
• • Left Homology Arm
    • Start: 238 End: 2978
  • Right Homology Arm
    • Start: 6269 End: 10600
• Misc. Feature (5 total)
• • loxP-1
    • Start: 3006 End: 3039
  • HSVTK-polyA
    • Start: 3046 End: 3304 (Complementary)
  • loxP-2
    • Start: 6212 End: 6245
• Promoter Eukaryotic (1 total)
• • Mus-PGK Promoter (correct)
    • Start: 5264 End: 5772 (Complementary)
• Replication Origin (2 total)
• • Replication Origin
    • Start: 10921 End: 11509 (Complementary)
Overview of Seq ID No. 45 (Downstream Vector, Step 2, Single Lox Feature Map
• CDS (3 total)
• • NEO (+STOP) CDS
    • Start: 3115 End: 3918 (Complementary)
  • TK CDS (from VEC1198)
    • Start: 3922 End: 5055 (Complementary)
  • AP(R)
    • Start: 11322 End: 12179 (Complementary)
    • bla gene—Ap(r) determinant
• Enhancer (1 total)
• • CMV Enhancer
    • Start: 5583 End: 6003 (Complementary)
• Misc. Binding Site (2 total)
• • Left Homology Arm
    • Start: 222 End: 2774
  • Right Homology Arm
    • Start: 6112 End: 10226
• Misc. Feature (4 total)
• • HSVTK-polyA
    • Start: 2850 End: 3108 (Complementary)
  • loxP-2
    • Start: 6016 End: 6049
• Promoter Eukaryotic (1 total)
• • Mus-PGK Promoter (correct)
    • Start: 5068 End: 5576 (Complementary)
• Replication Origin (2 total)
• • ORI
    • Start: 10511 End: 10511
    • RNaseH cleavage point
  • Replication Origin
    • Start: 10511 End: 11099 (Complementary)
Overview of Seq ID No. 46 (Upstream Vector Alternative, Step 2, Single Lox) Feature Map
• CDS (3 total)
• • NEO (+STOP) CDS
    • Start: 3311 End: 4114 (Complementary)
  • TK CDS (from VEC1198)
    • Start: 4118 End: 5251 (Complementary)
  • AP(R)
    • Start: 11698 End: 12555 (Complementary)
    • bla gene—Ap(r) determinant
• Enhancer (1 total)
• • CMV Enhancer
    • Start: 5779 End: 6199 (Complementary)
• Misc. Binding Site (2 total)
• • Left Homology Arm
    • Start: 238 End: 2978
  • Right Homology Arm
    • Start: 6235 End: 10566
• Misc. Feature (4 total)
• • loxP-1
    • Start: 3006 End: 3039
  • HSVTK-polyA
    • Start: 3046 End: 3304 (Complementary)
• Promoter Eukaryotic (1 total)
• • Mus-PGK Promoter (correct)
    • Start: 5264 End: 5772 (Complementary)
• Replication Origin (2 total)
• • ORI
    • Start: 10887 End: 10887
    • RNaseH cleavage point
  • Replication Origin
    • Start: 10887 End: 11475 (Complementary)
Overview of Seq ID No. 47 (Downstream Vector Alternative, Step 1, Double Lox) Feature Map
• CDS (3 total)
• • NEO (+STOP) CDS
    • Start: 3149 End: 3952 (Complementary)
  • TK CDS (from VEC1198)
    • Start: 3956 End: 5089 (Complementary)
  • AP(R)
    • Start: 11356 End: 12213 (Complementary)
    • bla gene—Ap(r) determinant
• Enhancer (1 total)
• • CMV Enhancer
    • Start: 5617 End: 6037 (Complementary)
• Misc. Binding Site (2 total)
• • Left Homology Arm
    • Start: 222 End: 2774
  • Right Homology Arm
    • Start: 6146 End: 10260
• Misc. Feature (5 total)
• • loxP-1
    • Start: 2844 End: 2877
  • HSVTK-polyA
    • Start: 2884 End: 3142 (Complementary)
  • loxP-2
    • Start: 6050 End: 6083
• Promoter Eukaryotic (1 total)
• • Mus-PGK Promoter (correct)
    • Start: 5102 End: 5610 (Complementary)
• Replication Origin (2 total)
• • Replication Origin
    • Start: 10545 End: 11133 (Complementary)
• The first vector pair is used to produce cells in which the entire J/cluster region is deleted.
• The second vector pair is used to produce cells in which the entire J/C cluster region is deleted.
Example 5: Crossbreeding of Heavy Chain Single Knockout with Kappa Single Knockout Pigs
• To produce pigs that have both one disrupted Ig heavy chain locus and one disrupted Ig light-chain kappa allele, single knockout animals were crossbred. The first pregnancy yielded four fetuses, two of which screened positive by both PCR and Southern for both heavy-chain and kappa targeting events (see examples 1 and 2 for primers). Fetal fibroblasts were isolated, expanded and frozen. A second pregnancy resulting from the mating of a kappa single knockout with a heavy chain single knockout produced four healthy piglets.
• Fetal fibroblast cells that contain a heavy chain single knockout and a kappa chain single knockout will be used for further targeting. Such cells will be used to target the lambda locus via the methods and compositions described herein. The resulting offspring will be heterozygous knockouts for heavy chain, kappa chain and lambda chain. These animals will be further crossed with animals containing the human Ig genes as described herein and then crossbred with other single Ig knockout animals to produce porcine Ig double knockout animals with human Ig replacement genes.
• This invention has been described with reference to its preferred embodiments. Variations and modifications of the invention, will be obvious to those skilled in the art from the foregoing detailed description of the invention.

Claims (1)

1. A targeting vector comprising: (a) a first nucleotide sequence comprising at least 17 contiguous nucleic acids homologous to SEQ ID No 28 or 31; (b) a selectable marker gene; and (c) a second nucleotide sequence comprising at least 17 contiguous nucleic acids homologous to SEQ ID No 28 or 31, which does not overlap with the first nucleotide sequence.

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