CN113355345B - Method for integrating exogenous sequences in genome - Google Patents

Abstract

The invention provides a method for genome integration of foreign sequences. The method comprises the following steps: 1) inserting the attB site corresponding to the site-specific recombinase Bxb1 on the genome of Eutropha rosenbergii to construct a recombinant Eutropha rosenbergii with the attB sequence integrated on the genome; Source sequence, site-specific recombinase Bxb1 gene, site-specific recombinase Bxb1 corresponding attP sequence recombinant vector; 3) the recombinant vector constructed in step 2) is transferred to the described recombinant vector constructed in step 1). In the recombinant E. rosenbergii, the site-specific recombinase Bxb1 is used to mediate the recombination between attB and attP sequences, so that the recombinant vector is integrated into the genome of the recombinant E. rosenbergii.

Description

A method for integrating exogenous sequences into genome

Technical Field

The invention belongs to the field of biotechnology and relates to a method for integrating an exogenous sequence into a genome, and in particular to a gene insertion method based on a site-specific recombinase applicable to Eutropha rhodesi.

Background Art

Ralstonia eutropha (also known as Cupriavidus necator) is an important model bacterium for studying the synthesis of PHA (polyhydroxyalkanoate, a fully degradable bio-based material) and has the potential to be used as a strain for the industrial production of PHA.

Existing gene editing techniques for R. rosea are based on homologous recombination using suicide plasmids as vectors. The suicide plasmid contains the R6Kγ replicon that cannot replicate in R. rosea, resistance genes, and DNA sequences for homologous recombination (called homologous arms), which can be freely designed according to the gene sites that need to be edited. When the suicide plasmid is transferred into R. rosea, under the selection pressure of antibiotics, homologous recombination occurs between the homologous arm sequences on the suicide plasmid vector and the same sequences on the genome, thereby achieving site-specific gene editing. In addition, a CRISPR/Cas9-based gene editing method has been developed in Ralstonia eutropha. The principle is also homologous recombination, but the selection pressure is changed to using Cas9 protein to cut the genome at a specific site (Xiong, B., Li, Z., Liu, L., Zhao, D., Zhang, X., Bi, C., 2018. Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique. Biotechnol. Biofuels. 11: 172).

Homologous recombination is the recombination between non-sister chromatids or between or within DNA molecules containing homologous sequences on the same chromosome. Homologous recombination depends on the presence of homologous regions or sequences. The breaks in the DNA chains are random, exposing some sequences that can bind to a series of proteins (such as RecA in prokaryotic cells and Rad51 in eukaryotic cells), ultimately achieving cross-recombination between or within DNA molecules. However, relying on random sequence breaks and the recombination system of the chassis microorganism itself will limit the length of the integrated exogenous sequence. In addition, it is necessary to optimize the homologous regions (such as the length and position of the homologous sequences) to achieve better gene editing effects.

Although homologous recombination can theoretically achieve gene knockout, insertion, and replacement, in actual applications, the larger the inserted fragment, the lower the efficiency of gene insertion, which limits its application in scenarios such as large-fragment gene insertion in multi-gene circuits, complex metabolic pathways, and so on.

In contrast, site-specific recombination does not rely on the homology of DNA sequences (although some are composed of very short homologous sequences) during the recombination process, but relies on the existence of short DNA sequences that can bind to certain enzymes. Under the action of these highly efficient and specific recombinases, the DNA chains break and reconnect. Therefore, it has site specificity, ensuring the high specificity and high conservatism of recombination. Site-specific recombination is also more efficient than homologous recombination methods that rely on proteins such as RecA.

The site-specific recombination system has many advantages such as high efficiency and controllability, accuracy and speed, strong specificity, and good orthogonality of elements. It has a wide range of application prospects and can be used in gene editing and genetic engineering operations. The system is derived from bacteriophages. When infecting bacteria, bacteriophages insert their DNA into the chromosomes of host bacteria and replicate and divide synchronously with the host. This process requires the site-specific recombinase in the bacteriophage to recognize special "recognition sites" - "attP" and "attB" (attP and attB exist in the phage and host chromosomes, respectively). The recombinase mediates the recombination between the two sequences of attP and attB. This process will insert the phage DNA into the host bacterial chromosome, and the original attP and attB sequences will form two new sequences "attL" and "attR" on the chromosome. As a tool, the site-specific recombination system consists of the recognition site/sequence of the integrase and the integrase that recognizes the DNA sequence and mediates DNA recombination. By changing the position and sequence direction of the recombinase recognition site, the site-specific recombinase can be used as a tool enzyme to insert a specific sequence into the DNA chain or delete or flip a specific sequence from the DNA chain. Therefore, it is widely used in different chassis microorganisms to perform any cutting and suturing operations (recombination of DNA fragments).

Site-specific recombination systems have been applied to different microorganisms and even eukaryotic cells, including Escherichia coli, Bacillus, Saccharomyces cerevisiae, T cells and even embryonic stem cells, and are widely used in the integration of exogenous genes, production of natural products and gene therapy, etc. For example, using integrase as a gene editing tool, the integration of a 34kb metabolic pathway in the Escherichia coli genome can be achieved for alginate degradation and ethanol production (Christine Nicole S. Santos., Drew D. Regitsky., and Yasuo Yoshikuni., 2013. Implementation of stable and complex biological systems through recombinase-assisted genome engineering. Nature Communications. 4: 2503).

In summary, the existing gene editing techniques for E. rosea are based on homologous recombination with suicide plasmids as vectors and CRISPR/Cas9-based gene editing methods. Homologous recombination and CRISPR/Cas9 gene editing methods become less efficient as the inserted fragment becomes longer when inserting genes. Site-specific recombinases can achieve efficient long-fragment insertion, but there are currently no site-specific recombinases and related tools available for E. rosea.

Summary of the invention

In view of the shortcomings of the prior art, the inventors have developed a gene insertion method based on site-specific recombinase suitable for Eutropha rosea, and combined with the existing homologous recombination technology, improved the gene editing technology of Eutropha rosea.

Therefore, in one aspect, the present invention provides a gene insertion method based on a site-specific recombinase suitable for Eutropha rosea, comprising the following steps:

1) inserting the attB site corresponding to the site-specific recombinase Bxb1 into the genome of E. rosenbergii to construct a recombinant E. rosenbergii with the attB sequence integrated into the genome;

2) constructing a recombinant vector containing an exogenous sequence, a site-specific recombinase Bxb1 gene, and an attP sequence corresponding to the site-specific recombinase Bxb1;

3) The recombinant vector constructed in step 2) is transferred into the recombinant Rhodesia eutropha constructed in step 1), and the site-specific recombinase Bxb1 is used to mediate the recombination between attB and attP sequences, thereby integrating the recombinant vector into the genome of the recombinant Rhodesia eutropha.

By the above method, the exogenous sequence (DNA fragment) can be integrated into the genome of the recombinant bacteria together with the vector. In practical applications, preferably, the vector part other than the DNA fragment to be integrated can be deleted as needed.

Therefore, the gene insertion method based on site-specific recombinase applicable to Eutropha rhodesi of the present invention preferably comprises the following steps:

a) inserting an attB site corresponding to the site-specific recombinase Bxb1 into the genome of E. rosenbergii to construct a recombinant E. rosenbergii with attB sequence integrated into the genome;

b) constructing a recombinant vector containing the VCre recombinase gene;

c) constructing a recombinant vector containing an exogenous sequence, a site-specific recombinase Bxb1 gene, an attP sequence corresponding to the site-specific recombinase Bxb1, and two VloxP sequences that can be specifically recognized by the VCre recombinase in step b), wherein the exogenous sequence and the attP sequence are between the two VloxP sequences, and the site-specific recombinase Bxb1 gene is not between the two VloxP sequences;

d) transferring the recombinant vector constructed in step c) into the recombinant R. roseae constructed in step a), and using the site-specific recombinase Bxb1 to mediate the recombination between attB and attP sequences, thereby integrating the recombinant vector into the genome of the recombinant R. roseae;

e) transferring the recombinant vector constructed in step b) into the recombinant bacteria obtained in step d) whose genome has been integrated with the recombinant vector, thereby deleting the backbone of the recombinant vector from the genome.

In step 1) or a) of the method of the present invention, the Ralstonia eutropha may also be other bacteria of the genus Ralstonia. Preferably, the Ralstonia eutropha may be Ralstonia eutropha H16.

In step 2) or c) of the method of the present invention, preferably, the recombinant vector may be a plasmid vector that cannot replicate in E. coli, such as a suicide plasmid. Preferably, the backbone portion of the recombinant vector comprises a replicon that can replicate in E. coli (such as S17-1) but cannot replicate in E. coli, preferably, the replicon is one or more selected from pMB1 replicon, pUC replicon, p15a replicon and R6Kγ replicon, more preferably, the replicon is pMB1 replicon. Preferably, the backbone portion of the recombinant vector also comprises a selection marker gene, such as an antibiotic resistance gene; more preferably, the backbone portion of the recombinant vector also comprises an antibiotic resistance gene, in particular, the antibiotic resistance gene is one or more selected from kanamycin resistance gene, tetracycline resistance gene, streptomycin resistance gene and spectinomycin resistance gene, more particularly, the antibiotic resistance gene is a kanamycin resistance gene. More preferably, the plasmid vector may be derived from plasmid pK18mobsacB.

In step b) of the method of the present invention, preferably, the recombinant vector may be a plasmid vector that can replicate in E. coli. Preferably, the backbone portion of the recombinant vector comprises a replicon that can replicate in E. coli (such as S17-1) and in E. coli. Preferably, the replicon is one or more selected from pBBR1 replicon, SC101 replicon and RK2 replicon, more preferably, the replicon is pBBR1 replicon. Preferably, the backbone portion of the recombinant vector also comprises a selection marker gene, such as an antibiotic resistance gene; more preferably, the backbone portion of the recombinant vector also comprises an antibiotic resistance gene, preferably, the antibiotic resistance gene is one or more selected from kanamycin resistance gene, tetracycline resistance gene, streptomycin resistance gene and spectinomycin resistance gene, more preferably, the antibiotic resistance gene is kanamycin and spectinomycin resistance gene. More preferably, the plasmid vector may be derived from plasmid pBBR1MCS2.

In step 1) or a) of the method of the present invention, preferably, the sequence of the attB site corresponding to the site-specific recombinase Bxb1 is as shown in SEQ ID NO:10.

In step 2) or c) of the method of the present invention, there is no particular requirement or limitation on the exogenous sequence. For example, the length of the exogenous sequence may be less than 1,000,000 bp, preferably less than 100,000 bp.

In step 2) or c) of the method of the present invention, preferably, the amino acid sequence of the site-specific recombinase Bxb1 gene is as shown in SEQ ID NO:20.

In step 2) or c) of the method of the present invention, preferably, the nucleotide sequence of the site-specific recombinase Bxb1 gene is as shown in SEQ ID NO:21.

In step 2) or c) of the method of the present invention, preferably, the attP sequence corresponding to the site-specific recombinase Bxb1 is as shown in SEQ ID NO:22.

In step b) of the method of the present invention, preferably, the amino acid sequence of the VCre recombinase is as shown in SEQ ID NO:42.

In step b) of the method of the present invention, preferably, the nucleotide sequence of the VCre recombinase is as shown in SEQ ID NO:41.

In step c) of the method of the present invention, preferably, the VloxP sequence is shown as SEQ ID NO:44.

In step 3) of the method of the present invention, preferably, the recombinant vector is transferred into Escherichia coli (e.g., S17-1), and then transferred into recombinant Roebuck eutropha by conjugation transformation, and the recombinant Roebuck eutropha with the recombinant vector integrated into the genome is screened out using a screening marker by utilizing the property that the suicide plasmid cannot replicate in the host bacteria.

Further preferably, step 1) or a) of the method of the present invention can be implemented by methods known in the art (Xiong, B., Li, Z., Liu, L., Zhao, D., Zhang, X., Bi, C., 2018. Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique. Biotechnol. Biofuels. 11: 172). For example, a suicide plasmid in which the attB site is located between two homologous fragments and cannot be replicated in Ralstonia eutropha is constructed, and the suicide plasmid is transferred into Ralstonia eutropha, and the characteristics that the suicide plasmid cannot be replicated in the host bacteria are used to obtain a recombinant Ralstonia eutropha with integrated attB sequence by screening. Specifically, step 1 or a) of the method of the present invention comprises the following steps: using SEQ ID NO: 1 and SEQ ID NO: 2 as primers to perform PCR amplification with the Ralstonia eutropha H16 genome as a template to obtain homologous fragments H1 and H2, using SEQ ID NO: 3 and SEQ ID NO: 4 as primers to perform PCR amplification with the plasmid pK18mobsacB as a template to obtain a vector fragment, using SEQ ID NO: 7 and SEQ ID NO: 8 as primers to perform PCR amplification with SEQ ID NO: 9 as a template to obtain a fragment containing the attB sequence corresponding to Bxb1, connecting H1, H2 and attB sequences with the vector fragment by the Gibson Assembly method, wherein the attB site is located between the two homologous fragments H1 and H2, to obtain a recombinant plasmid pK18mobsacB-Bxb1; transferring the recombinant plasmid pK18mobsacB-Bxb1 into Escherichia coli S17-1, and then transferring it into Ralstonia eutropha by a conjugation transformation method. In H16, the suicide plasmid is unable to replicate in the host bacteria, and the positive clones are screened using LB plates containing both kanamycin and apramycin. The recombinant plasmid with homologous fragments in the positive clones is integrated into the specific positions of H1 and H2 on the genome, which is the first homologous recombinant bacteria; the first homologous recombinant bacteria are plated out and cultured as single clones on LB plates containing sucrose, and clones without kanamycin resistance are screened from these single clones, and the recombinant bacteria in which the attB sequence of Bxb1 is integrated into the genome are identified, and the obtained recombinant bacteria are Ralstonia eutropha Bxb1-attB.

Further preferably, step 2) of the method of the present invention includes the following steps: using SEQ ID NO: 5 and SEQ ID NO: 6 as primers to amplify the vector fragment using plasmid pK18mobsacB as a template, using SEQ ID NO: 9 and SEQ ID NO: 10 as primers to amplify the fragment containing the Bxb1 recombinase gene and its corresponding attP sequence using SEQ ID NO: 19 as a template, and connecting the fragment with the vector fragment by the Gibson Assembly method to obtain the recombinant plasmid pBxb1-attP.

Further preferably, step 3) of the method of the present invention comprises the following steps: transferring the recombinant plasmid pBxb1-attP into Escherichia coli (such as S17-1), and then transferring it into Ralstonia eutropha Bxb1-attB by conjugation transformation, utilizing the property that the suicide plasmid cannot replicate in the host bacteria, and simultaneously screening by screening markers to obtain recombinant Ralstonia eutropha with the recombinant vector integrated into the genome.

Further preferably, step b) of the method of the present invention comprises the following steps: using SEQ ID NO:36 and SEQ ID NO:37 as primers and plasmid pBBR1MCS2 as a template to PCR amplify to obtain a replicon fragment; using SEQ ID NO:38 and SEQ ID NO:39 as primers and SEQ ID NO:40 as a template to amplify to obtain a DNA fragment, which contains a VCre recombinase gene, a kanamycin resistance gene and a spectinomycin resistance gene; connecting the fragment with the replicon fragment by the Gibson Assembly method to obtain a recombinant plasmid pVCre.

Further preferably, step c) of the method of the present invention comprises the following steps: using SEQ ID NO: 5 and SEQ ID NO: 6 as primers to obtain a vector fragment by PCR amplification with plasmid pK18mobsacB as a template; obtaining a fragment containing the Bxb1 recombinase gene and its corresponding attP sequence, the exogenous gene to be integrated, and two VloxP sequences that can be specifically recognized by the VCre recombinase, and connecting the fragment with the vector fragment by the Gibson Assembly method to obtain a recombinant plasmid. Specifically, taking GFP as an example, step c) of the method of the present invention comprises the following steps: using SEQ ID NO:5 and SEQ ID NO:6 as primers to obtain a vector fragment by PCR amplification with plasmid pK18mobsacB as a template; using SEQ ID NO:17 and SEQ ID NO:18 as primers to obtain a DNA fragment by amplification with SEQ ID NO:43 as a template, wherein the fragment contains the Bxb1 recombinase gene and its corresponding attP sequence, the exogenous gene to be integrated (GFP), and two VloxP sequences that can be specifically recognized by VCre recombinase; and connecting the fragment with the vector fragment by the Gibson Assembly method to obtain the recombinant plasmid pBxb1-attP-VCre.

Further preferably, step d) of the method of the present invention comprises the following steps: transferring the recombinant plasmid of step c), such as pBxb1-attP-VCre, into Escherichia coli S17-1, and then transferring it into Ralstonia eutrophaBxb1-attB by conjugation transformation, and obtaining recombinant Ralstonia eutropha with the recombinant vector integrated into its genome by screening with a screening marker.

Further preferably, step e) of the method of the present invention comprises the following steps: transferring the recombinant plasmid pVCre obtained in step b) into Escherichia coli S17-1, transferring it into the recombinant bacteria of step d) by conjugation transformation method, and obtaining the recombinant bacteria (e.g. Ralstonia eutropha Bxb1-GFP) in which the exogenous gene is integrated into the genome by screening through screening markers.

Preferably, step e) of the method of the present invention further comprises: culturing the recombinant bacteria with the exogenous gene integrated into the genome on a non-resistant plate to cause the recombinant bacteria to lose the recombinant vector containing the VCre recombinase gene.

In the gene insertion method based on site-specific recombinase applicable to Eutropha rosea of the present invention, as shown in FIG1: first, an attB site corresponding to the site-specific recombinase Bxb1 is inserted into the genome of Eutropha rosea to construct a recombinant Eutropha rosea with an attB sequence integrated into the genome; a recombinant vector containing a VCre recombinase gene is constructed; a recombinant vector containing an exogenous sequence, a site-specific recombinase Bxb1 gene, an attP sequence corresponding to the site-specific recombinase Bxb1, and two VloxP sequences that can be specifically recognized by the above-mentioned VCre recombinase is constructed, wherein the exogenous sequence and the attP sequence are between the two VloxP sequences, and the site-specific recombinase Bxb1 gene is not between the two VloxP sequences (as shown in FIG1). 2), and the recombinant vector is transferred into the recombinant Rhodesia eutropha with attB sequence integrated into the genome constructed above, and the site-specific recombinase Bxb1 is used to mediate the recombination between attB and attP sequences, so that the recombinant vector is integrated into the genome of the recombinant Rhodesia eutropha, wherein the recombinase mediates the recombination between attP and attB sequences, and this process will insert the phage DNA into the host bacterial chromosome, and the original attP and attB sequences will form two new sequences "attL" and "attR" on the chromosome; then, the recombinant vector containing the VCre recombinase gene constructed above is transferred into the recombinant bacteria with the recombinant vector integrated into the genome, so that the backbone part of the recombinant vector is deleted from the genome.

On the other hand, the present invention provides a recombinant vector (suicide plasmid), which comprises an exogenous sequence, a site-specific recombinase Bxb1 gene, and an attP sequence corresponding to the site-specific recombinase Bxb1. Preferably, the recombinant vector further comprises two VloxP sequences that can be specifically recognized by the VCre recombinase, wherein the exogenous sequence and the attP sequence are between the two VloxP sequences, and the site-specific recombinase Bxb1 gene is not between the two VloxP sequences. Preferably, the backbone portion of the recombinant vector comprises a replicon that can replicate in Escherichia coli (such as S17-1) but cannot replicate in Rhodesia eutropha, preferably, the replicon is one or more selected from pMB1 replicon, pUC replicon, p15a replicon and R6Kγ replicon, more preferably, the replicon is pMB1 replicon. The backbone of the recombinant vector further comprises an antibiotic resistance gene, preferably, one or more of a kanamycin resistance gene, a tetracycline resistance gene, a streptomycin resistance gene and a spectinomycin resistance gene, more preferably, the antibiotic resistance gene is a kanamycin resistance gene. Preferably, the amino acid sequence of the site-specific recombinase Bxb1 gene is as shown in SEQ ID NO:20. More preferably, the nucleotide sequence of the site-specific recombinase Bxb1 gene is as shown in SEQ ID NO:21. Preferably, the attP sequence corresponding to the site-specific recombinase Bxb1 is as shown in SEQ ID NO:22. Preferably, the VloxP sequence is as shown in SEQ ID NO:44.

In another aspect, the present invention provides a recombinant vector comprising a VCre recombinase gene. Preferably, the backbone portion of the recombinant vector comprises a replicon that can replicate in Escherichia coli (such as S17-1) and in Eutropha rhodesiae, preferably, the replicon is selected from one or more of pBBR1 replicon, SC101 replicon and RK2 replicon, more preferably, the replicon is pBBR1 replicon. The backbone portion of the recombinant vector also comprises an antibiotic resistance gene, preferably, comprising one or more of a kanamycin resistance gene, a tetracycline resistance gene, a streptomycin resistance gene and a spectinomycin resistance gene, more preferably, the antibiotic resistance gene is a kanamycin and a spectinomycin resistance gene. Preferably, the amino acid sequence of the VCre recombinase is shown in SEQ ID NO:42. More preferably, the nucleotide sequence of the VCre recombinase is shown in SEQ ID NO:41.

The invention develops a site-specific recombinase tool available in Ralstonia eutropha, which can realize efficient integration of exogenous sequences into the genome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the site-specific recombinase-mediated gene integration process of the present invention.

FIG. 2 is a schematic diagram of the vector structure containing an exogenous sequence, a recombinase and an attP site in the present invention.

FIG. 3 is a diagram showing the PCR results for verifying the integration of the vector into the genome mediated by the recombinase Bxb1 in Example 3 of the present invention.

FIG. 4 is a diagram showing the PCR results for verifying vector backbone deletion in Example 4 of the present invention.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

The enzyme reagents used were purchased from ThermoFisher and New England Biolabs (NEB), the kit used to extract the plasmid was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the kit for recovering DNA fragments was purchased from Omega, USA. The corresponding operation steps were carried out strictly in accordance with the product instructions, and all culture media were prepared with deionized water unless otherwise specified.

Culture medium formula:

LB medium: 5 g/L yeast extract (purchased from OXID, UK, product catalog number LP0021), 10 g/L peptone (purchased from OXID, UK, product catalog number LP0042), 10 g/L NaCl, and the rest is water. Adjust the pH value to 7.0-7.2 and sterilize with high pressure steam.

In the actual culture process, a certain concentration of antibiotics may be added to the above culture medium to maintain the stability of the plasmid, such as 200 μg/mL kanamycin, 100 μg/ml apramycin or 500 μg/mL spectinomycin.

Example 1: Construction of recombinant Ralstonia eutropha Bxb1-attB integrating attB sequence

Master Mix，购买自New England Biolabs(NEB)公司)的说明，将H1、H2和attB序列通过Gibson Assembly方法与载体片段连接，得到重组质粒pK18mobsacB-Bxb1。使用的引物如下表：The homologous fragments H1 and H2 were obtained by PCR amplification using the genome of Ralstonia eutropha H16 (purchased from China General Microbiological Culture Collection Center, CGMCC1.7092) as a template. The vector fragment was obtained by PCR amplification using the plasmid pK18mobsacB (Orita, I., Iwazawa, R., Nakamura, S., Fukui, T., 2012. Identification of mutation points in Cupriavidus necator NCIMB 11599 and genetic reconstitution of glucose-utilization ability in wild strain H16 for polyhydroxyalkanoate production. J. Biosci. Bioeng. 113, 63-69) as a template. The fragment containing the attB sequence corresponding to Bxb1 was amplified using primers using the synthetic fragment 01 as a template. The commercial kit (Gibson

Master Mix, purchased from New England Biolabs (NEB) company), was connected to the vector fragment by Gibson Assembly method to obtain the recombinant plasmid pK18mobsacB-Bxb1. The primers used are as follows:

The sequence of synthetic fragment 01 is:

GGCAGAGAGACAATCAAATCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCCAGGAAACAGCTATGACGGTTCGGCCGGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCATCCGGGCACTGGCCGTCGTTTTACAACCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTC CATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTATTGGTGAGAATCCAGCCTGCCGGCCTGGTTCAAC(SEQ ID NO:9)

The attB sequence corresponding to Bxb1 is:

TCGGCCGGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCATCCGGGC(SEQ ID NO:10)

The recombinant plasmid pK18mobsacB-Bxb1 was transferred into Escherichia coli S17-1 (ATCC No.: 47055, available from American Type Culture Collection), and then transferred into Ralstonia eutropha H16 by conjugation transformation. The suicide plasmid cannot replicate in the host bacteria, and positive clones were screened using LB plates containing 200 μg/ml kanamycin and 100 μg/ml apramycin. The recombinant plasmid with homologous fragments in the positive clone was integrated into the specific positions of H1 and H2 on the genome, which was the first homologous recombinant bacteria.

The first homologous recombinant bacteria were streaked out as single clones on LB plates containing 100 mg/ml sucrose, and clones without kanamycin resistance were screened from these single clones. PCR was performed using primers primer 7 and primer 8 to identify the recombinant bacteria in which the attB sequence of Bxb1 was integrated into the genome. The resulting recombinant bacteria were Ralstonia eutropha Bxb1-attB.

Comparative Example 1: Construction of recombinant Ralstonia eutropha PhiC31-attB with integrated attB sequence

Master Mix，购买自New England Biolabs(NEB)公司)的说明，将H1、H2和attB序列通过Gibson Assembly方法与载体片段连接，得到重组质粒pK18mobsacB-PhiC31。使用的引物如下表：The homologous fragments H1 and H2 were obtained by PCR amplification using the Ralstonia eutropha H16 genome as a template, the vector fragment was obtained by PCR amplification using the plasmid pK18mobsacB as a template, and the fragment containing the attB sequence corresponding to PhiC31 was amplified using primers and synthetic fragment 02 as a template. The fragment was PCR-amplified according to the commercial kit (Gibson

Master Mix, purchased from New England Biolabs (NEB) company), was connected to the vector fragment by Gibson Assembly method to obtain the recombinant plasmid pK18mobsacB-PhiC31. The primers used are as follows:

The sequence of synthetic fragment 02 is:

GGCAGAGAGACAATCAAATCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCCAGGAAACAGCTATGACGGTCGCGCCCGGGGAGCCCAAGGGCACGCCCTGGCACACTGGCCGTCGTTTTACAACCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAAC GCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGCCTGCCGGCCTGGTTCAAC(SEQ ID NO:11)

The attB sequence corresponding to PhiC31 is:

CGCGCCCGGGGAGCCCAAGGGCACGCCCTGGCAC(SEQ ID NO:12)

The recombinant plasmid pK18mobsacB-PhiC31 was transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha H16 by conjugation transformation. The suicide plasmid could not replicate in the host bacteria, and the positive clones were screened using LB plates containing 200 μg/ml kanamycin and 100 μg/ml apramycin. The recombinant plasmid with homologous fragments in the positive clone was integrated into the specific positions of H1 and H2 on the genome, which was the first homologous recombination bacteria.

The first homologous recombinant bacteria were streaked out as single clones on LB plates containing 100 mg/ml sucrose, and clones without kanamycin resistance were screened from these single clones. PCR was performed using primers primer 7 and primer 8 to identify the recombinant bacteria in which the attB sequence of PhiC31 was integrated into the genome. The resulting recombinant bacteria were Ralstonia eutropha PhiC31-attB.

Comparative Example 2: Construction of recombinant Ralstonia eutropha TP901-attB with integrated attB sequence

Master Mix，购买自New England Biolabs(NEB)公司)的说明，将H1、H2和attB序列通过Gibson Assembly方法与载体片段连接，得到重组质粒pK18mobsacB-TP901。使用的引物如下表：The homologous fragments H1 and H2 were obtained by PCR amplification using the Ralstonia eutropha H16 genome as a template, the vector fragment was obtained by PCR amplification using the plasmid pK18mobsacB as a template, and the fragment containing the attB sequence corresponding to TP901 was amplified using primers and synthetic fragment 03 as a template. The fragment was PCR amplified according to the commercial kit (Gibson

Master Mix, purchased from New England Biolabs (NEB) company), was connected to the vector fragment by Gibson Assembly method to obtain the recombinant plasmid pK18mobsacB-TP901. The primers used are as follows:

The sequence of synthetic fragment 03 is:

GGCAGAGAGACAATCAAATCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCCAGGAAACAGCTATGACGGTTATGCCAACACAATTAACATCTCAATCAAGGTAAATGCTTTTTGCTTTTTTACTGGCCGTCGTTTTACAACCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCAT CTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTATTGGTGAGAATCCAGCCTGCCGGCCTGGTTCAAC(SEQ ID NO:13)

The attB sequence corresponding to TP901 is:

TATGCCAACACAATTAACATCTCAATCAAGGTAAATGCTTTTTGCTTTTTTTG(SEQ ID NO:14)

The recombinant plasmid pK18mobsacB-TP901 was transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha H16 by conjugation transformation. The suicide plasmid could not replicate in the host bacteria, and the positive clones were screened using LB plates containing 200 μg/ml kanamycin and 100 μg/ml apramycin. The recombinant plasmid with homologous fragments in the positive clone was integrated into the specific positions of H1 and H2 on the genome, which was the first homologous recombination bacteria.

The first homologous recombinant bacteria were streaked out as single clones on LB plates containing 100 mg/ml sucrose, and clones without kanamycin resistance were screened from these single clones. PCR was performed using primers primer 7 and primer 8 to identify the recombinant bacteria in which the attB sequence of TP901 was integrated into the genome. The resulting recombinant bacteria were Ralstonia eutropha TP901-attB.

Comparative Example 3: Construction of recombinant Ralstonia eutropha P22-attB integrating attB sequence

Master Mix，购买自NewEngland Biolabs(NEB)公司)的说明，将H1、H2和attB序列通过Gibson Assembly方法与载体片段连接，得到重组质粒pK18mobsacB-P22。使用的引物如下表：The homologous fragments H1 and H2 were obtained by PCR amplification using the Ralstonia eutropha H16 genome as a template, the vector fragment was obtained by PCR amplification using the plasmid pK18mobsacB as a template, and the fragment containing the attB sequence corresponding to P22 was amplified using primers and synthetic fragment 04 as a template. The fragment was PCR-amplified according to the commercial kit (Gibson

Master Mix, purchased from New England Biolabs (NEB) Company), was used to connect H1, H2 and attB sequences to the vector fragment by Gibson Assembly method to obtain the recombinant plasmid pK18mobsacB-P22. The primers used are as follows:

The sequence of synthetic fragment 04 is:

GGCAGAGAGACAATCAAATCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCCAGGAAACAGCTATGACGGTACGACCTTCGCATTACGAATGCGCTGCACTGGCCGTCGTTTTACAACCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGG TTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGCCTGCCGGCCTGGTTCAAC(SEQ ID NO:15)

The attB sequence corresponding to P22 is:

ACGACCTTCGCATTACGAATGCGCTGC(SEQ ID NO:16)

The recombinant plasmid pK18mobsacB-P22 was transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha H16 by conjugation transformation. The suicide plasmid could not replicate in the host bacteria, and the positive clones were screened using LB plates containing 200 μg/ml kanamycin and 100 μg/ml apramycin. The recombinant plasmid with homologous fragments in the positive clone was integrated into the specific positions of H1 and H2 on the genome, which was the first homologous recombination bacteria.

The first homologous recombinant bacteria were streaked out as single clones on LB plates containing 100 mg/ml sucrose, and clones without kanamycin resistance were screened from these single clones. PCR was performed using primers primer 7 and primer 8 to identify the recombinant bacteria in which the attB sequence of P22 was integrated into the genome. The resulting recombinant bacteria were Ralstonia eutropha P22-attB.

Example 2: Construction of a recombinant plasmid pBxb1-attP containing a recombinase and the corresponding attP sequence

Master Mix，购买自New England Biolabs(NEB)公司)的说明，将片段通过Gibson Assembly方法与载体片段连接，得到重组质粒pBxb1-attP。使用的引物如下表：Plasmid pK18mobsacB was used as a template for PCR amplification to obtain a vector fragment. The synthetic fragment 05 was used as a template to amplify a fragment containing the Bxb1 recombinase gene and its corresponding attP sequence. The fragment was cloned according to a commercial kit (Gibson

Master Mix, purchased from New England Biolabs (NEB) Company), was connected to the vector fragment by Gibson Assembly method to obtain the recombinant plasmid pBxb1-attP. The primers used are as follows:

The sequence of synthetic fragment 05 is:

CACACAGGAAACAGCTATGACCTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCTACGACATCCCGGTGTGTAGCCGTTCGACCACGCTGCCGAGCCTGAGATGCTGCTCGTACTCTTGCAGATCCCCGAAGTCGATCGTGCGAGTCAGCCCGCCGCGGACGTCGAACGTCAGCCGAACGTTCATCG ACCGAAGCCAGGTGTTCTTTGCCGCGGTGTCCTGCTCCCGCCACCAGTCCCCGAACCGCTGCCCGGTCTCCGCCACTCCCAGCCAGACGGGCGAGCCTCTAGGCCCTCCAGCTCCTCTTGCCGCGCGGCCAGCGCCCGCAATACGGGCATCCAGTGCTTCTCGCTGCGGAGAGCCGGCCCGGTAGGCCGGGGAGCCGATCAGCG ACGTCAGGTCCACCAGCTCCGCGTTCACCTCCGCGAGTTCGACCGCGGAGTCCGAGCCGGCTACCCAGACTTTCTCCAGACGCTCCGCGTCCCCGAGCAGATCCAGCACCTGCTCCTCCGCAGAACGCGTCCCACTCGGCCATCGCCACCGTGCCGTTCCCGCAGTGCTTCGGGAACCCCATCGAGCGGCAGCGGTAGCGCGGGTGCTTACGTCCTCCCCCGGCGAACTTGTACGCGGGCTCCCCGCACACCGCGCA GAACAACACCCGCAGCAGCAGCGACGGGGTAGACACCGCGGGCTTCGCCCGGGAGGTCTCTCACGAGCTCGGCGCGCAGCGCCTCCAGCTGCTCACGGGTCAGGATCGGCTCAGCCCGCACCAGCGGGGCTCCGTCGTCGTCTCGGACGGTCTTACCGTTCAGAGTCGCGTACCCGAGCATCGCCTCGGAGATCATCGATCGCTTCA GCGCGGTAGCCGACCACTCCCGGCCCTGCGGCTCGCGGCCTTGCAGCTGCGCGAAGTAGTCCTTCGGCGACAGGACACCACGCCGGTTCAGGTCGTGGGCCACCAGGTGCAGCGGCTCGTGGTTGTCGACGACGCGGTGATACACCTCGAGGATGCGCTCTCGCTGCACAGGGTCCGGCACCAGCCGCCACTCCCCGTCCACGCGCGTAGGCAGGTATCCCCACGGCGGCAGGGATCCTCGGTATTTCCC GGCGCGGATATTGAAATGCGCAGCCGAACGGTTCCGCTCTTTGATCGCTTCTAATTCCATCTGCGCCACCGTTCCCATAAGCGCGATGACGACCGCCGCAAACGGCGTCGTCGTATCGAAGTGCGCTTCGGTCGCGGAGACGACCAGCTTCTTGTGGTCCTCGGCCCAGTGGACCAGCTGTTGCAGATGCCGGATCGATCGGGTCAACCGGT CTACCCGGTACGCCACGATCACGTCGAACGGTTGCTCCTCGAACGCTAGCCACCGGGCCAGGTTCGGTCTGCGCTTCCGGTCGAACGGATCGACCGCCCCGGAGACGTCCAGATCCTCCGCTACCCCGACGACGTCCCAGCCGCGCTGGGCGCAGAGCTGCTGGCAAGACTCCAGCTGACGCTCCGGTGAAGTCGTAGCATCGGTGACGCGGGACAGGCGGATGACTACCAGGGCTCTCATCTAGTATTTCTCC TCTTTCTCTAGTATTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCCGCAGGTCAAAGGGTATACTGGGATTCCAGTGAACGCAAGGGTTTGTACCGTACACCACTGAGACCGCGGTGGTTGACCAGACAAACCACGAACTGGCCGTCGTTTTACAAC(SEQ ID NO:19)

The gene sequence of Bxb1 recombinase is:

ATGAGAGCCCTGGTAGTCATCCGCCTGTCCCGCGTCACCGATGCTACGACTTCACCGGAGCGTCAGCTGGAGTCTTGCCAGCCTCTGCGCCCAGCGCGGCTGGGACGTCGTCGGGGTAGCGGAGGATCTGGACGTCTCCGGGGCGGTCGATCCGTTCGACCGGAAGCGCAGACCGAACCTGGCCCGGTGGCTAGCGTTCGAGGAGCAACCGTTCGACGTGATCGTGGCGTACCGGGTAGACCGGTTGACCC GATCGATCCGGCATCTGCAACAGCTGGTCCACTGGGCCGAGGACCACAAGAAGCTGGTCGTCTCCGCGACCGAAGCGCACTTCGATACGACGACGCCGTTTGCGGCGGTCGTCATCGCGCTTA TGGGAACGGTGGCCGCAGATGGAATTAGAAGCGATCAAAGAGCGGAACCGTTCGGCTGCGCATTTCAATATCCGCGCCGGGAAATACCGAGGATCCCTGCCGCCGTGGGGATACCTGCCTACGCGCGTGGACGGGGAGTGGCGGCTGGTGCCGGACCCTGTGCAGCGAGAGCGCATCCTCGAGGTGTATCACCGCGTCGTCGACAACCACGAGCCGCTGCACCTGGTGGCCCACCGACCTGAACCGGCGTGGT GTCCTGTCGCCGAAGGACTACTTCGCGCAGCTGCAAGGCCGCGAGCCGCAGGGCCGGGAGTGGTCGGCTACCGCGCTGAAGCGATCGATGATCTCCGAGGCGATGCTCGGGTACGCGACTCTGAAC GGTAAGACCGTCCGAGACGACGACGGAGCCCCGCTGGTGCGGGCTGAGCCGATCCTGACCCGTGAGCAGCTGGAGGCGCTGCGCGCCGAGCTCGTGAAGACCTCCCGGGCGAAGCCCGCGGTGTCTACCCCGTCGCTGCTGCTGCGGGTGTTGTTCTGCGCGGTGTGCGGGGAGCCCGCGTACAAGTTCGCCGGGGGAGGACGTAAGCACCCGCGCTACCGCTGCCGCTCGATGGGGTTCCCGAAGCACTGC GGGAACGGCACGGTGGCGATGGCCGAGTGGGACGCGTTCTGCGAGGAGCAGGTGCTGGATCTGCTCGGGGACGCGGAGCGTCTGGAGAAAGTCTGGGTAGCCGGCTCGGACTCCGCGGTCGAACT CGCGGAGGTGAACGCGGAGCTGGTGGACCTGACGTCGCTGATCGGCTCCCCGGCCTACCGGGCCGGCTCTCCGCAGCGAGAAGCACTGGATGCCCGTATTGCGGCGCTGGCCGCGGCAAGAGGAGCTGGAGGGCCTAGAGGCTCGCCCGTCTGGCTGGGAGTGGCGCGAGACCGGGCAGCGGTTCGGGGACTGGTGGCGGGAGCAGGACACCGCGGCAAAGAACACCTGGCTTCGGTCGATGAACGTTC GGCTGACGTTCGACGTCCGCGGCGGGCTGACTCGCACGATCGACTTCGGGGATCTGCAAGAGTACGAGCAGCATCTCAGGCTCGGCAGCGTGGTCGAACGGCTACACACCGGGATGTCGTAG (SEQ ID NO: 20)

The amino acid sequence of the Bxb1 recombinase is:

MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLDVSGAVDPFDRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLVVSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGYLPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPK DYFAQLQGREPQGREWSATALKRSMISEAMLGYATLNG KTVRDDDGAPLVRAEPILTREQLEALRAELVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTVAMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRAGSPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSMNVRLTFDVRGGLTRTIDFG DLQEYEQHLLRGSVVERLHTGMS(SEQ ID NO:21)

The attP sequence corresponding to Bxb1 is:

TCGTGGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAACCC (SEQ ID NO: 22) Comparative Example 4: Construction of a recombinant plasmid pPhiC31-attP containing a recombinase and the corresponding attP sequence

Master Mix，购买自New England Biolabs(NEB)公司)的说明，将片段通过Gibson Assembly方法与载体片段连接，得到重组质粒pPhiC31-attP。使用的引物如下表：Plasmid pK18mobsacB was used as a template for PCR amplification to obtain a vector fragment. The synthetic fragment 06 was used as a template to amplify a fragment containing the PhiC31 recombinase gene and its corresponding attP sequence. The fragment was amplified according to a commercial kit (Gibson

Master Mix, purchased from New England Biolabs (NEB) Company), was connected to the vector fragment by Gibson Assembly method to obtain the recombinant plasmid pPhiC31-attP. The primers used are as follows:

The sequence of synthetic fragment 06 is:

CACACAGGAAACAGCTATGACCTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCTACGCCGCTACGTCTTCCGTGCCGTCCTGGGCGTCGTCTTCGTCGTCGTCGGTCGGCGGCTTCGCCCACGTGATCGAAGCGCGCTTCTCGATGGGCGTTCCCTGCCCCCTGCCCTGTAGTCGACTTCGTGACAA CGATCTTGTC TACGAAGAGCCCGACGAACACGCGCTTGTCGTCTACTGACGCGCGCCCACCACCACGACTTAGGGCCGGTCGGGTCAGCGTCGGCGTCTTCGGGGAACCATTGGTCAAGGGGAAGCTTCGGGGCTTCGGCGGCTTCAAGTTCGGCAAGCCGCTCTTCCGCCCCTTGCTGCCGGAGCGTCAGCGCTGCCTGTTGCTTCCGGAAGTGCTTCCTGCCAACGGGTCCGTCGTACGCGCCTGCCGCGGCGTCTTCGT ACAGCTCTTCAAGGGCGT TCAGGGCGTCGGCGCTCCGCAACAAGGTTCGCCCGTTCGCCGCTTCTCAGGCGCCTCAGTGAGCTTGCCGAAGCGTCGGGCGGCTTCCCACAGAAGCGCCAACGTCTCTTCGTCGCCTTCGGCGTGCCTGATCTTGTTGAAGATGCGTTCCGCAACGAACTTGTCGAGTGCCGCCATGCTGACGTTGCACGTGCCTTCGTGCTGCCCAGGTGCGGACGGGTCGACCACCTTCCGGCGACGGCAGCGGTA AGAGTCCTTGATCGA TTCTTCCCCGCGCTTCGAAGTCATGACGGCCGCCACACTCGCAGTACAGCTTGTCCATGGCGGACAGAATGGCTCGCCCGGGAAAGCCCCTTGCCGCGCCCCCTGCCGTCCAACCACGCCTGAAGCTCATACCACTCAGCGGGCTCGATGATCGGTCCGCAATCAAGCTCGACCGGCCGGAGCGTGATCGGGTCGCGCTGAATGCGGTAACCCTCAATCTCGTGGTCGGCGTGCCGTCCGGCTTCTTCTTG TAGATCACCTCAGGCGG CGAAGCCCGCAATACGCGGGTCCCGAAGGATTCGCATAACGGTTGCCGGGTCCCAGGCGCTTGAAGCGGTCTTCTTCCCAATCGTCTCGCCCCGGGTCGGCACGGCGTCAGCGTCCATGCGCTTACAAAGCCCCGTGATGCTGCCCGGGTGAATGGCGGCTTGACTGCCCGGCTTGAAGGGAAGGTGTTTGCGTCTTGATCTCACGCCACCACCGGATTACGTCGGGCTCGAACTCGAAGGGTCCGGTAAG GGGAGTGGTCGA GTGCGCAAGCTTGTTGATGACGACATTGACCATTCGGCCGTTGCGCGTGATCTCCTTCGTCTCCGAAACAAGCTCGAAGCCGTAAGGCGCCTTCCCGCCGACGTACCCGCCCAATTCGCGCTGAAGGTTCTTCGTGTCGAGAATCTTCGCCGACTTCAGCGAAGATTCTTTGTGCGACGCGTCGAGCCGCATAATCAGGTGAATCAGGTCCATGACGTTTCCCTGCCGGAAGACGCCTTCCTGAGTGGAAAAAA TCGTCACGCCCAGGG CGAGCAATTCCGAGACAATCGGAATCGCGTCCATGACCTTCAGGCGCGAGAAGCGCGACACGTCATAGACAATGATCATGTTGAGCCGCCCGGCGCGGCATTCGTTCAGGATGCGTTCGAACTCCGGGCGCTCCGCCGTCCCGAACGCCGACGTGCCCGGCGCTTCGCTGAAATGCCCGACGAACCTGAACCGGCCCCCGTCGCGCTCGACTTCGCGCTGAAGGTCGGCCGCCTTGTCTTCGTTGGCGCTACG CTGTGTCGCTGGGCTT GCTGCGCTCGAATTCTCGCGCTCGCGCGACTGACGGTCGTAAGCACCCGCGTACGTGTCCATCTAGTATTTCTCCTTTTCTCTAGTATTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCGCAGGTCAAAGGGTATACTGGGATTCCAGTGAACGCAACCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGACTGGCCGTCGTTTT ACAAC(SEQ ID NO:23)

The gene sequence of PhiC31 recombinase is:

ATGGACACGTACGCGGGTGCTTACGACCGTCAGTCGCGCGAGCGCGAGAATTCGAGCGCAGCAAGCCCAGCGACACAGCGTAGCGCCAACGAAGACAAGGCGGCCGACCTTCAGCGCGAAGTCGAGCGCGACGGGGGCCGGTTCAGGTTCGTCGGGCATTTCAGCGAAGCGCCGGGCACGTCGGCGTTCGGGACGGCGGAGCGCCCGGAGTTCGAACGCATCCTGAACGAATGCCGCGCCGGGCGGCTCAACAT GATCATTGTCTATGACGTGTCGCGCTTCTCGCGCCTGAAGGTCATGGACGCGATTCCGATTGTCTCGGAATTGCTCGCCCTGGGCGTGACGATTGTTTCCACTCAGGAAGGCGTCTTCCGGCAGGGAAACGTCATGGACCTGATTCACCTGATTATGCGGCTCGACGCGTCGCACAAAGAATCTTCGCTGAAGTCGGCGAA GATTCTCGACACGAAGAACCTTCAGCGCGAATTGGGCGGGTACGTCGGCGGGAAGGCGCCTTACGGCTTCGAGCTTGTTTCGGAGACGAAGGAGATCACGCGCAACGGCCGAATGGTCAATGTCGTCATCAACAAGCTTGCGCACTCGACCACTCCCCTTACCGGACCCTTCGAGTTCGAGCCCGACGTAATCCGGTGGTGGTGGCGTGAGATCAAGACGCACAAACACCTTCCCTTCAAGCCGGGCAGTCAAGCCGCCATTCA CCCGGGCAGCATCACGGGGCTTTGTAAGCGCATGGACGCTGACGCCGTGCCGACCCGGGGCGAGACGATTGGGAAGAAGACCGCTTCAAGCGCCTGGGACCCGGCAACCGTTATGCGAATCCTTCGGGACCCGCGTATTGCGGGCTTCGCCGCTGAGGTGATCTACAAGAAGAAGCCGGACGGCACGCCGAC CACGAAGATTGAGGGTTACCGCATTCAGCGCGACCCGATCACGCTCCGCCGTCGAGCTTGATTGCGGACCGATCATCGAGCCCGCTGAGTGGTATGAGCTTCAGGCGTGGTTGGACGGCAGGGGGCGCGGCAAGGGGCTTTCCCGGGGGCAAGCCATTCTGTCCGCCATGGACAAGCTGTACTGCGAGTGTGGCGCCGTCATGACTTCGAAGCGCGGGGAAGAATCGATCAAGGACTCTTACCGCTGCCGTC GCCGGAAGGTGGTCGACCCGTCCGCACCTGGGCAGCACGAAGGCACGTGCAACGTCAGCATGGCGGCACTCGACAAGTTCGTTGCGGAACGCATCTTCAACAAGATCAGGCACGCCGAAGGCGACGAAGAGACGTTGGCGCTTCTGTGGGAAGCCGCCCGACGCTTCGGCAAGCTCACTGAGGCGCCTGAGAAGAGCGGCG AACGGGCGAACCTTGTTGCGGAGCGCGCCGACGCCCTGAACGCCCTTGAAGAGCTGTACGAAGACCGCGCGGCAGGCGCGTACGACGGACCCGTTGGCAGGAAGCACTTCCGGAAGCACAGGCAGCTGACCGGCAGCAAGGGGCGGAAGAGCGGCTTGCCGAACTTGAAGCCGCCGAAGCCCCGAAGCTTCCCCTTGACCAATGGTTCCCCGAAGACGCCGACGCTGACCCGACCGGCCCTAAGTCGTGGTG GGGGCGCGCGTCAGTAGACGACAAGCGCGTGTTCGTCGGGCTCTTCGTAGACAAGATCGTTGTCACGAAGTCGACTACGGGCAGGGGGCAGGGAACGCCCATCGAGAAGCGCGCTTCGATCACGTGGGCGAAGCCGCCGACCGACGACGACGAAGACGACGCCCAGGACGGCACGGAAGACGTAGCGGCGTAG (SEQ ID NO: 24)

The amino acid sequence of the PhiC31 recombinase is:

MDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDGGRFRFVGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIPIVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQRELGGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWREIKTHK HLPFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRILRDPRIAGFAAEVIYKKKPDGTPT TKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQAWLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPGQHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERANLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAEAPKLP LDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPIEKRASITWAKPPTDDDEDDAQDGTEDVAA(SEQ ID NO:25)

The attP sequence corresponding to PhiC31 is:

CCCCCAACTGAGAGAACTCAAAGGTTACCCCAGTTGGGG(SEQ ID NO:26)

Comparative Example 5: Construction of a recombinant plasmid pTP901-attP containing a recombinase and corresponding attP sequence

Master Mix，购买自New England Biolabs(NEB)公司)的说明，将片段通过Gibson Assembly方法与载体片段连接，得到重组质粒pTP901-attP。使用的引物如下表：Plasmid pK18mobsacB was used as a template for PCR amplification to obtain a vector fragment. The synthetic fragment 07 was used as a template for amplification to obtain a fragment containing the TP901 recombinase gene and its corresponding attP sequence. The fragment was cloned according to a commercial kit (Gibson

Master Mix, purchased from New England Biolabs (NEB) Company), was connected to the vector fragment by Gibson Assembly method to obtain the recombinant plasmid pTP901-attP. The primers used are as follows:

The sequence of synthetic fragment 07 is:

CACACAGGAAACAGCTATGACCTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGTTAAGCAGCCAGAGCGTAGTTTTCGTCCTTAGCAGCACCGGTAGCGAGTTGGAATTTAAATATGATATCTACATTATCAGCAGTAACATCAACCTTTGATACAAGGTTGTTGACGATTTTCTTTTTATTCATATG ATAGTTCATTAATCGGAATTGAGCCCAACTGAGTTTTAACTAACTCAAAAACATCAGTAGAGTCATTAAATTTATTTTCGCTAATCTTAGCTTTAAGCAGCTTTTCTCCAGCCTGAAGGGAATCAGTACGATCTTTCAACTCATCCATAGTGATAAAATCATTTAGGTACAAATCAGAGTTCTTTTGTATTTTTTTATCGATCTGT GAAATTTGCTTTTTAAATGACGAAGTATCAAGAATAGGTTGGTTGTTGCCATTGATAATTTTCAATAAGGAGTCATTATTTTCTTGAAATCCAATCAGGTTGTCAATAACAGTATTTTCTAAATTACTTAAATCATAAGTTCCTGAATCACACTTTTTATTGTCATTATATACTGTAATTCCTTTTGTTTTTCGAGGAAATCTATTTGCACAGTGATATTTCATAGTGCGGCTTCCATCTTTTCTTTTGTGGCCAAGAACAATT TTTAAAGGTGCTCCACAGTAACCGCACCTTGCCATCCCTGACAGCATATATTTAGCTTGGAAAGGTCTAGGGTTGTTATTTCTTTCATAAGTCTGCTGTTGTCTTTCTTCTAGCTCTTTTTGAACTTTAAATAAGTCTCATAAGGGATAATTGGTTTGTGCATACCTTCAAATAGGCTGTCCTTAAATTTGATATAACCACA GTAAACTGGATTATCAAGTGTTTGTCTTAGGGTACGATAAGACCACGGTATATCTTACCGATGTGTCCAGATTCATTGAGTTTATCTCCTTAATTTTGTAAGTGATATTCCTGATAAATAATCAGTGAATATTTGTTCAACTATTGTAGCTTGTAAAGGAACAATTTCTAATATACCTGTCTTTCTGTTGTGGTAATACCCAAAAGCTGTCTTAGTCCACATCATAGACTTACCAGATTTCGCTCGCCCTAGTTTACCCATA GTCATGCGTTCTTTTATATTCTCTTTCAAACTCATTAATTGCAGAAAGAATAGTGAGAAACAAGCTACCCATAGCAGAAGAAGTATCAATACTTTCATTAAGCGAGATAAAGTCTATTTTATTTTTTGTGAACACATCCTTAACAAGATAAAGAGTATCTCTTACACTACGTGAAAGGCGGTCTAGCTTATATACAAGAACTG TATCAAAAGCTTTATTCTCGATATCGTTGATTAATCTTTGCATTGCTGGGCGTTCAAGTTTGGCCCCTGAAAAACCAGCATCAGTATAAGTATCAGATACTTGCCACCCCATTGCTTCAGCATATTTTGTTAAACGGTCAATTTGCTCATCAATTGAGAAGCCTTCCTCTGCTTGGTTAGTAGTGGATACTCGTGTATAGATTGCTACTTTCTTAGTGCCGGCCTGGTGGTGATGGTGATGATGTTTCATTCTAGTATTTC TCCTCTTTCTCTAGTATTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCGCAGGTCAAAGGGTATACTGGGATTCCAGTGAACGCAAAAAAGGAGTTTTTTAGTTACCTTAATTGAAATAAACGAAATAAAAACTCGACTGGCCGTCGTTTTACAAC (SEQ ID NO: 27)

The gene sequence of TP901 recombinase is:

ATGAAACATCATCACCATCACCACCAGGCCGGCACTAAGAAAGTAGCAATCTATACACGAGTATCCACTACTAACCAAGCAGAGGAAGGCTTCTCAATTGATGAGCAAATTGACCGTTTAACAAAATATGCTGAAGCAATGGGGTGGCAAGTATCTGATACTTATACTGATGCTGGTTTTTCAGGGGCCAAACTTGAACGCCCAGCAATGCAAAGATTAATCAACGATATCGAGAATAAAGCTTTTGATACAGTTCTTGTATATA AGCTAGACCGCCTTTCACGTAGTGTAAGAGATACTCTTTATTCTTGTTAAGGATGTGTTCACAAAAAATAAAATAGACTTTATCTCGCTTAATGAAAGTATTGATACTTCTTCTGCTA TGGGTAGCTTGTTTCTCACTATTCTTTCTGCAATTAATGAGTTTGAAAGAGAGAATATAAAAGAACGCATGACTATGGGTAAACTAGGGCGAGCGAAATCTGGTAAGTCTATGATGTGGACTAAGACAGCTTTTGGGTATTACCACAACAGAAAGACAGGTATATTAGAAATTGTTCCTTTACAAGCTACAATAGTTGAACAAATATTCACTGATTATTTATCAGGAATATCACTTACAAAATTAAGAGATAAACTCAATGAAT CTGGACACATCGGTAAAGATATACCGTGGTCTTATCGTACCCTAAGACAAACACTTGATAATCCAGTTTACTGTGGTTATATCAAATTTAAGGACAGCCTATTTGAAGGTATGCACAAA CCAATTATCCCTTATGAGACTTATTTAAAAGTTCAAAAAGAGCTAGAAGAAAGACAACAGCAGACTTATGAAAGAAATAACAACCCTAGACCTTTCCAAGCTAAATATATGCTGTCAGGGATGGCAAGGTGCGGTTACTGTGGAGCACCTTTAAAAATTGTTCTTGGCCACAAAAGAAAAGATGGAAGCCGCACTATGAAATATCACTGTGCAAATAGATTTCCTCGAAAAACAAAAGGAATTACAGTATATAATGACAATAAAAAGTG TGATTCAGGAACTTATGATTTAAGTAATTTAGAAATACTGTTATTGACAACCTGATTGGATTTCAAGAAAATAATGACTCCTTATTGAAAATTATCAATGGCAACAACCAACC TATTCTTGATACTTCGTCATTTAAAAAGCAAATTTCACAGATCGATAAAAAAATACAAAAGAACTCTGATTTGTACCTAAATGATTTTATCACTATGGATGAGTTGAAAGATCGTACTGATTCCCTTCAGGCTGAGAAAAAGCTGCTTAAAGCTAAGATTAGCGAAAATAAATTTAATGACTCTACTGATGTTTTTGAGTTAGTTAAAACTCAGTTGGGCTCAATTCCGATTAATGAACTATCATATGATAAAAAGAAAATCGTCAA CAACCTTGTATCAAAGGTTGATGTTACTGCTGATAATGTAGATATCATATTTAAATTCCAACTCGCTACCGGTGCTGCTAAGGACGAAAACTACGCTCTGGCTGCTTAA(SEQ ID NO:28)

The amino acid sequence of TP901 recombinase is:

MKHHHHHHQAGTKKVAIYTRVSTTNQAEEGFSIDEQIDRLTKYAEAMGWQVSDTYTDAGFSGAKLERPAMQRLINDIENKAFDTVLVYKLDRLSRSVRDTLYLVKDVFTKNKIDFISLNESIDTSSAMGSLFLTILSAINEFERENIKERMTMGKLGRAKSGKSMMWTKTAFGYYHNRKTGILEIVPLQATIVEQIFTDYLSGISLTKLRDKLNES GHIGKDIPWSYRTLRQTLDNPVYCGYIKFKDSLFEGMHKP IIPYETYLKVQKELEERQQQTYERNNNPRPFQAKYMLSGMARCGYCGAPLKIVLGHKRKDGSRTMKYHCANRFPRKTKGITVYNDNKKCDSGTYDLSNLENTVIDNLIGFQENNDSLLKIINGNNQPILDTSSFKKQISQIDKKIQKNSDLYLNDFITMDELKDRTDSLQAEKKLLKAKISENKFNDSTDVFELVKTQLGSIPINELSYD NKKKIVNNLVSKVDVTADNVDIIFKFQLATGAAKDENYALAA(SEQ ID NO:29)

The attP sequence corresponding to TP901 is:

CGAGTTTTTATTTCGTTTATTTCAATTAAGGTAACTAAAAAACTCCTTTT (SEQ ID NO: 30) Comparative Example 6: Construction of a recombinant plasmid pP22-attP containing a recombinase and a corresponding attP sequence

Master Mix，购买自New England Biolabs(NEB)公司)的说明，将片段通过Gibson Assembly方法与载体片段连接，得到重组质粒pP22-attP。使用的引物如下表：Similarly, plasmid pK18mobsacB was used as a template for PCR amplification to obtain a vector fragment, and the synthetic fragment 08 was used as a template for amplification to obtain a fragment containing the P22 recombinase gene and its corresponding attP sequence. The fragment was cloned according to the commercial kit (Gibson

Master Mix, purchased from New England Biolabs (NEB) Company), was connected to the vector fragment by Gibson Assembly method to obtain the recombinant plasmid pP22-attP. The primers used are as follows:

The sequence of synthetic fragment 08 is:

CACACAGGAAACAGCTATGACCTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCTACGTATTATTCGTGCCTTCCTTATTTTTACTGTGGGACATATTTGGGACAGAAGTACCAAAAATCGAGTCAATTTGTCGAGCATGTTCAGTCAGGTGATTTGGTGCCAGATGAGCATTCGGCGAACCATTTCGATAG ACTCCCAGCCACCCATTTCCTGCAATACCGAAATCGGAACGCCAGCCTGAACTAACCAACTTGCCCACGTGTGCCTCAGGTCATGAAAACGGAAGTCTTCAATGCCCGCTCGTTTTAATGCTGCCCTCCATGCAGTATTAGCGTCATAGCGCATCTTCCTCACTA CAGGTGATTTAGTTCCGTCTGGTTTGGTGCTGCTTTCCTTGTAGACGAACACCCATTTGTGATGATTGCCGATTTGCTTTTTCAGCACCCGGCAAGCGGTATCATTCAGCGCCACTCCAATGGCATGATTAGACTTGCTTTGTTCCGGGTGTATCCATGCCACCTTTCGTTGCATGTCTATCTGCTGCCACTCCAGATTGATAATGTTAGACCGCCTTAAGCCAGTAGAAAGCGCAAACTCTACGACTGACTTTAGC GGTTCCTGGCATTCATCAATCAACCTTTTTGCCTCGTGAGGCTCAAGCCAGCGGATACGCTTATTTTTCGGCTGAGGAACTTTGATGATCGGAGCCTTATCCAGCATCTTCCATTCGCGTTCAGCAGCCCGGAGGAGTGCCTTAATGAATGAAAGGTGAGTTGCTTTTGTAG CTACTGCTGCCGGCTTAGGCTTGAATACCGGAGGCTGCTTCCCATTCTTCCTGCATGCTTCATCCATTAACTTCCAGTTTTCCTCATGCCGCCGATTAGTTATTCTTCTGGATGGCGGAGTAAATCTTCGTCTCGGTAATATCCTTCAACTGCATTCCTGCAAAATGCTGGAGCCAGAATCCTATCCGACTCTTGTCATCATCCAGCGACTTCTTATGCGCCTTCTCCTCTAACCACCTGACACAGGCCCCCTCAAAAGT CATGTCAGGCGTCTCTCCTAATTTACTTACCCTCCATGCTTCTGCCTTCAGCTTGTCATGAAGCTCTGTGGCCTGCCTTTTGTCCTTTGTCCCAAGAGACTGCTTAAATCTTTTGCCGTTCGGCAATGTGAAACTGGCGTACCAGGTTTCACCTCTGCGGAATAGTGACA TCTAGTATTTCTCCTTCTCTAGTATTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCCGCAGGTCAAAGGGTATACTGGGATTCCAGTGAACGCAACTAAGTGGTTTGGGACAAAAATGGGACATACAAATCTTTGCATCGGTTTGCAAGGCTTTGCATGTCTTTCGAAGATGGGACGTGTGAGCGCAGGTATGACGTGGTATGTTGT TGACTTAAAAGGTAGTTCTTATAATTCGTAATGCGAAGGTCGTAGGTTCGACTCCTATTATCGGCACCAGTTAAATCAAATACTTACGTATTATTCGTGCCTTCCTTATTTTTACTGTGGGACATATTTGGGACAGAAGTACCAAAAAACTGGCCGTCGTTTTACAAC (SEQ ID NO: 31)

The gene sequence of P22 recombinase is:

ATGTCACTATTCCGCAGAGGTGAAACCTGGTACGCCAGTTTCACATTGCCGAACGGCAAAAGATTTAAGCAGTCTCTTGGGACAAAGGACAAAAGGCAGGCCACAGAGCTTCATGACAAGCTGAAGGCAGAAGCATGGAGGGTAAGTAAATTAGGAGAGACGCCTGACATGACTTTTGAGGGGGCCTGTGTCAGGTGGTTAGAGGAGAAGGCGCATAAGAAGTCGCTGGATGATGACAAGAGTCGGATAGGATTCTGGCTCC AGCATTTTGCAGGAATGCAGTTGAAGGATA TTACCGAGACGAAGATTTACTCCGCCATCCAGAAGATAACTAATCGGCGGCATGAGGAAAACTGGAAGTTAATGGATGAAGCATGCAGGAAGAATGGGAAGCAGCCTCCGGTATTCAAGCCTAAGCCGGCAGCAGTAGCTACAAAAGCAACTCACCTTTCATTCATTAAGGCACTCCTCCGGGCTGCTGAACGCGAATGGAAGATGCTGGATAAGGCTCCGATCATCAAAGTTCCTCAGCCGAAAAAGCGTATCCGCT GGCTTGAGCCTCACGAGGCAAAAAGGTTGAT TGATGAATGCCAGGAACCGCTAAAGTCAGTCGTAGAGTTTGCGCTTTCTACTGGCTTAAGGCGGTCTAACATTATCAATCTGGAGTGGCAGCAGATAGACATGCAACGAAAGGTGGCATGGATACACCCGGAACAAGTCTAATCATGCCATTGGAGTGGCGCTGAATGATACCGCTTGCCGGGTGCTGAAAAAGCAAATCGGCAATCATCACAAATGGGTGTTCGTCTACAAGGAAAGCAGCACCAAACCAGACG GAACTAAATCACCTGTAGTGAGGAAGATGCGC TATGACGCTAATACTGCATGGAGGGCAGCATTAAAACGAGCGGGCATTGAAGACTTCCGTTTTCATGACCTGAGGCACACGTGGGCAAGTTGGTTAGTTCAGGCTGGCGTTCCGATTTCGGTATTGCAGGAAATGGGTGGCTGGGAGTCTATCGAAATGGTTCGCCGATATGCTCATCTGGCACCAAATCACCTGACTGAACATGCTCGACAAATTGACTCGATTTTTGGTACTTCTGTCCCAAATATGTCCCACAGTAAAAATA AGGAAGGCACGAATAATACGTAG(SEQ ID NO:32)

The amino acid sequence of the P22 recombinase is:

MSLFRRGETWYASFTLPNGKRFKQSLGTKDKRQATELHDKLKAEAWRVSKLGETPDMTFEGACVRWLEEKAHKKSLDDDKSRIGFWLQHFAGMQLKDITETKIYSAIQKITNRRHEENWKLMDEACRKNGKQPPVFKPKPAAVATKATHLSFIKALLRAAEREWKMLDKAPIIKVPQPKNKRIRWLEPHEAKRLIDECQEPLK SVVEFALSTGLRRSNIINLEWQQIDMQRKVAWIHPEQSKSNHAIGVALNDTACRVLKKQIGNHHKWVFVYKESSTKPDGTKSPVVRKMRYDANTAWRAALKRAGIEDFRFHDLRHTWASWLVQAGVPISVLQEMGGWESIEMVRRYAHLAPNHLTEHARQIDSIFGTSVPNMSHSKNKEGTNNT(SEQ ID NO:33)

The attP sequence corresponding to P22 is:

TTTTTGGTACTTCTGTCCCAAATATGTCCCACAGTAAAAATAAGGAAGGCACGAATAATACGTAAGTATTTGATTTAACTGGTGCCGATAATAGGAGTCGAACCTACGACCTTCGCATTACGAATTATAAGAACTACCTTTTAAGTCAACAACATACCACGTCATACCTGCGCTCACACGTCCCATCTTCGAAAGACATGCAAAGCCTTGCAAACCGATGCAAAGATTTGTATGTCCCATTTTTGTCCCAAACCACTTAG (SEQ ID NO: 34)

Example 3: Recombinase-mediated attB-attP recombination

The recombinant plasmid pBxb1-attP was transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha Bxb1-attB by conjugation transformation. The suicide plasmid cannot replicate in the host bacteria, and the LB plate containing 200 μg/ml kanamycin and 100 μg/ml apramycin was used for screening. If the recombinase Bxb1 is functional, it will mediate the recombination between attB and attP sequences, thereby integrating the plasmid pBxb1-attP into the genome.

Comparative Example 7: Recombinase-mediated attB-attP recombination

The recombinant plasmid pPhiC31-attP was transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha PhiC31-attB by conjugation transformation. The suicide plasmid cannot replicate in the host bacteria, and the LB plate containing 200 μg/ml kanamycin and 100 μg/ml apramycin was used for screening. If the recombinase PhiC31 is functional, it will mediate the recombination between attB and attP sequences, thereby integrating the plasmid pPhiC1-attP into the genome.

Comparative Example 8: Recombinase-mediated attB-attP recombination

The recombinant plasmid pTP901-attP was transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha TP901-attB by conjugation transformation. The suicide plasmid cannot replicate in the host bacteria, and the LB plate containing 200 μg/ml kanamycin and 100 μg/ml apramycin was used for screening. If the recombinase TP901 is functional, it will mediate the recombination between attB and attP sequences, thereby integrating the plasmid pTP901-attP into the genome.

Comparative Example 9: Recombinase-mediated attB-attP recombination

The recombinant plasmid pP22-attP was transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha P22-attB by conjugation transformation. The suicide plasmid cannot replicate in the host bacteria, and the LB plate containing 200 μg/ml kanamycin and 100 μg/ml apramycin was used for screening. If the recombinase P22 is functional, it will mediate the recombination between attB and attP sequences, thereby integrating the plasmid pP22-attP into the genome.

Experimental Example 1: Verification of vector integration into the genome

After the plasmids of the four recombinases were respectively transferred into the corresponding Ralstonia eutropha, 8 clones grown were randomly selected and PCR verification was performed using primers gcgcatggcgtctccatg (SEQ ID NO: 35) and gtggaccagctgttgcag (SEQ ID NO: 36). The results are shown in the following table:

The results in the above table show that only the attB and attP recombination mediated by the recombinase Bxb1 obtained the expected 862 bp band, proving that the plasmid pBxb1-attP was integrated into the genome (Figure 3), while the attB and attP recombination mediated by the recombinases PhiC31, TP901 and P22 were unsuccessful.

It can be seen that there is no inevitability or predictability whether a recombinase that has been proven to be useful in some host microorganisms will function in another microorganism. Among the four recombinases tested in the present invention, only Bxb1 has functioned in Ralstonia eutropha and can be used for the integration of exogenous sequences; while the other three recombinases have no function and are not suitable for use in Ralstonia eutropha strains.

Example 4: Deletion of the vector backbone using a helper plasmid

The above examples prove that the recombinase Bxb1 is functional in Ralstonia eutropha. Placing the DNA fragment to be integrated on a vector containing the Bxb1 gene and the corresponding attP sequence can achieve integration of the DNA fragment and the vector into the genome of the recombinant bacterium Ralstonia eutropha Bxb1-attB. In practical applications, more preferably, the vector part other than the DNA fragment to be integrated needs to be deleted. This example uses another set of recombinases to achieve this purpose.

Master Mix，购买自New England Biolabs(NEB)公司)的说明，将片段通过Gibson Assembly方法与复制子片段连接，得到重组质粒pVCre。使用的引物如下表：Plasmid pBBR1MCS2 (Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM., 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene. 166, 175-176) was used as a template for PCR amplification to obtain a replicon fragment; primers were used to amplify the synthetic fragment 09 as a template to obtain a DNA fragment containing the VCre recombinase gene, the kanamycin resistance gene and the spectinomycin resistance gene; the commercial kit (Gibson

Master Mix, purchased from New England Biolabs (NEB) Company), was connected to the replicon fragment by the Gibson Assembly method to obtain the recombinant plasmid pVCre. The primers used are as follows:

The sequence of synthetic fragment 09 is:

GAGCCAGCCGGTGGCCGCCTACATGGCTCTGCTGTAGTTCACCCTTGGCGTCCAACCAGCGGCACCAGCGGCGCCTGAGAGGGGCGCGCCCAGCTGTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTTTAATTAAAGCGGATAACAATTTCACACAGGACAACTGAGACCGGAATTGGTCTCAACGTACG TCTCATTTTCGCCAGATATCGACGTCTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAA GGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGT ACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAG CTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCTAGACATCATTAATTCCTAATTTTTGTTGACACTCTATCGTTGATAGA GTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGAATTCAAGCTGTCACCGGATTGCTTTCCGGTCTGATGAGTCCGTGAGG ACGAAACAGCCTCTACAAATAATTTTGTTTAATACTAGAGAAAGAGGAGAAATACTAGATGATCGAGAACCAGCTGAGCCTGCTGGGTGATTTCAGCGGCGTGCGTCCGGACGATGTTAAGACCGCGATCCAGGCGGCGCAAAAGAAAGGTATTAACGTTGCGGAGAACGAACAATTCAAAGCGGCGTTTGAGCACCTGCTGAACGAGTTCAAGAAACGTGAGGAACGTTACAGCCCGAACACCCTGCGTCGTCTGG AAAGCGCGTGGACCTGCTTTGTGGATTGGTGCCTGGCGAACCATCGTCACAGCCTGCCGGCGACCCCGGACACCGTTGAGGCGTTCTTTAT CGAACGTGCGGAGGAACTGCACCGTAACACCCTGAGCGTGTACCGTTGGGCGATTAGCCGTGTTCATCGTGTTGCGGGTTGCCCGGACCCGTGCTGGATATCTATGTGGAGGATCGTCTGAAGGCGATTGCGCGTAAGAAAGTGCGTGAGGGCGAAGCGGTTAAACAGGCGAGCCCGTTTAACGAACAACACCTGCTGAAGCTGACCAGCCTGTGGTACCGTAGCGACAAACTGCTGCTGCGTCGTAACCTGGC GCTGCTGGCGGTGGCGTATGAGAGCATGCTGCGTGCGAGCGAACTGGCGAACATCCGTGTTAGCGACATGGAGCTGGCGGGTGATGGCACCG CGATTCTGACCATCCCGATTACCAAGACCAACCACAGCGGCGAGCCGGACACCTGCATTCTGAGCCAGGATGTGGTTAGCCTGCTGATGGACTACACCGAAGCGGGCAAGCTGGACATGAGCAGCGATGGTTTCCTGTTTGTGGGCGTTAGCAAACACAACACCTGCATCAAGCCGAAGAAAGATAAACAGACCGGTGAAGTTCTGCACAAGCCGATTACCACCAAAACCGTGGAGGGCGTTTTCTATAGCGCGTGGGGAAACCCTG GATCTGGGTCGTCAAGGCGTGAAGCCGTTTACCGCGCACAGCGCGCGTGTTGGTGCGGCGCAGGACCTGCTGAAGAAAGGCT ACAACACCCTGCAAATCCAGCAAAGCGGTCGTTGGAGCAGCGGCGCGATGGTTGCGCGTTATGGTCGTGCGATCCTGGCGCGTGACGGCGCGATGGCGCACAGCCGTGTGAAAACCCGTAGCGCGCCGATGCAATGGGGCAAGGACGAGAAAGATTAATGATAAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTACTAGAGTC ACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGAGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCG CTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTACTAGAGGTCATGCTTGCCATCTGTTTTCTTGCAAGATTACTAGTAGCGGCCGCTGCAGGTCGTGACTGGGAAAACCCTGGCGACTAGTCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTT TTATTGGTGAGAATCCAGACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTG TCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACG GAATTTATGCCTCTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAAC AGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCCTGGCATGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCA GTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGG AATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATC AGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCAT CAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCTATTCTGACCTTGCCATCACGACTGTGCTGGTCATTAAACGCGTATTCAGGCTGACCCTGCGCGCTGCGCAGGGCTTTATTGATTCCATTTTTACACTGATGAATGTTCCGTTGCGCTGCCCGGATTACAGCCGGATCCTTCTAGAGTCGACCTGCAGGCATG CTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCT AAGGAAGCTAAAATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGTGATCGCCGAAGT ATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGA TGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAG CCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGA TCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCT CGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAAACTAGTAAATAATAAAAAA GCCGGATTAATAATCTGGCTTTTTATATTCTCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTTTTTCGGTATATCCATCCTTTTTCGCACGATATACAGGATTTTGCCAAAGGGTTCGTGTAGACTTTCCTTGGTGTATCCAACGGCGTCAGCCGGGCAGGATAGGTGAAGTAGGCCCACCCGCGAGCGGGTGTTCCTTCTTCACTGTCCCTTATTCGCACCTGGCGGTGCTCAACGGGAATCCTGCTCTGCGAGGC TGGCCGTAGGCCGGCCGCGATGCAGGTGGCTGCTGAACCCCCAGCCGGAACTGACCCCACAAGGCCCTACCGGCGCGGCAGCG (SEQ ID NO: 41)

The gene sequence of VCre recombinase is:

ATGATCGAGAACCAGCTGAGCCTGCTGGGTGATTTCAGCGGCGTGCGTCCGGACGATGTTAAGACCGCGATCCAGGCGGCGCAAAAGAAAGGTATTAACGTTGCGGAGAACGAACAATTCAAAGCGGCGTTTGAGCACCTGCTGAACGAGTTCAAGAAACGTGAGGAACGTTACAGCCCGAACACCCTGCGTCGTCTGGAAAGCGCGTGGACCTGCTTTGTGGATTGGTGCCTGGCGAACCATCGTCACAGCCTGCCGGC GACCCCGGACACCGTTGAGGCGTTCT TTATCGAACGTGCGGAGGAACTGCACCGTAACACCCTGAGCGTGTACCGTTGGGCGATTAGCCGTGTTCATCGTGTTGCGGGTTGCCCGGACCCGTGCCTGGATATCTATGTGGAGGATCGTCTGAAGGCGATTGCGCGTAAGAAAGTGCGTGAGGGCGAAGCGGTTAAACAGGCGAGCCCGTTTAACGAACAACACCTGCTGAAGCTGACCAGCCTGTGGTACCGTAGCGACAAACTGCTGCTGCGTCGTAACC TGGCGCTGCTGGCGGTGGCGTATGAGAGCATG CTGCGTGCGAGCGAACTGGCGAACATCCGTGTTAGCGACATGGAGCTGGCGGGTGATGGCACCGCGATTCTGACCATCCCGATTACCAAGACCAACCACAGCGGCGAGCCGGACACCTGCATTCTGAGCCAGGATGTGGTTAGCCTGCTGATGGACTACACCGAAGCGGGCAAGCTGGACATGAGCAGCGATGGTTTCCTGTTTGTGGGCGTTAGCAAACACAACACCTGCATCAAGCCGAAGAAAGATAAACAGACCGG TGAAGTTCTGCACAAGCCGATTACCAC CAAAACCGTGGAGGGCGTTTTCTATAGCGCGTGGGAAACCCTGGATCTGGGTCGTCAAGGGCTGAAGCCGTTTACCGCGCACAGCGCGCGTGTTGGTGCGGCGCAGGACCTGCTGAAGAAAGGCTACAACACCCTGCAAATCCAGCAAAGCGGTCGTTGGAGCAGCGGCGCGATGGTTGCGCGTTATGGTCGTGCGATCCTGGCGCGTGACGGCGCGATGGCGCACAGCCGTGTGAAAACCCGTAGCGCGCCGATGCAA TGGGGCAAGGACGAGAAAGATTAA(SEQ ID NO:42)

The amino acid sequence of VCre recombinase is:

MIENQLSLLGDFSGVRPDDVKTAIQAAQKKGINVAENEQFKAAFEHLLNEFKKREERYSPNTLRRLESAWTCFVDWCLANHRHSLPATPDTVEAFFIERAEELHRNTLSVYRWAISRVHRVAGCPDPCLDIYVEDRLKAIARKKVREGEAVKQASPFNEQHLLKLTSLWYRSDKLLLRRNLALLAVAYESMLRASELANIRVSDMELAGDGTAILTIP ITKTNHSGEPDTCILSQDVVSSLLMDYTEAGKLDMSSDGFLFVGVSKHNTCIKPKKDKQTGEVLHKPITTKTVEGVFYSAWETLDLGRQGVKPFTAHSARVGAAQDLLKKGYNTLQIQQSGRWSSGAMVARYGRAILARDGAMAHSRVKTRSAPMQWGKDEKD(SEQ ID NO:43)

Master Mix，购买自New England Biolabs(NEB)公司)的说明，将片段通过Gibson Assembly方法与载体片段连接，得到重组质粒pBxb1-attP-VCre。使用的引物如下表：The vector fragment was amplified by PCR using plasmid pK18mobsacB as a template; the DNA fragment was amplified using primers and synthetic fragment 10 as a template, which contained the Bxb1 recombinase gene and its corresponding attP sequence, the foreign gene to be integrated (green fluorescent protein gene GFP in this embodiment), and two VloxP sequences specifically recognized by VCre recombinase; the commercial kit (Gibson

Master Mix, purchased from New England Biolabs (NEB) Company), was connected to the vector fragment by Gibson Assembly method to obtain the recombinant plasmid pBxb1-attP-VCre. The primers used are as follows:

The sequence of synthetic fragment 10 is:

CACACAGGAAACAGCTATGACCTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCTACGACATCCCGGTGTGTAGCCGTTCGACCACGCTGCCGAGCCTGAGATGCTGCTCGTACTCTTGCAGATCCCCGAAGTCGATCGTGCGAGTCAGCCCGCCGCGGACGTCGAACGTCAGCCGAACGTTCATCG ACCGAAGCCAGGTGTTCTTTGCCGCGGTGTCCTGCTCCCGCCACCAGTCCCCGAACCGCTGCCCGGTCTCGCGCCACTCCCAGCCAGACGGGCGAGCCT CTAGGCCCTCCAGCTCCTCTTGCCGCGGCCAGCGCCCGCAATACGGGCATCCAGTGCTTCTCGCTGCGGAGAGCCGGCCCGGTAGGCCGGGGAGCCGATCAGCGACGTCAGGTCCACCAGCTCCGCGTTCACCTCCGCGAGTTCGACCGCGGAGTCCGAGCCGGCTACCCAGACTTTCTCCAGACGCTCCGCGTCCCCGAGCAGATCCAGCACCTGCTCCTCGCAGAACGCGTCCCACTCGGCCATCGCCACC GTGCCGTTCCCGCAGTGCTTCGGGAACCCCATCGAGCGGCAGCGGTAGCGCGGGTGCTTACGTCCTCCCCCGGCGAACTTGTACGCGGGCTCCCCGCACACCG CGCAGAACAACACCCGCAGCAGCGACGGGGTAGACACCGCGGGCTTCGCCCGGGAGGTCTTCACGAGCTCGGCGCGCAGCGCCTCCAGCTGCTCACGGGTCAGGATCGGCTCAGCCCGCACCAGCGGGGCTCCGTCGTCGTCTCGGACGGTCTTACCGTTCAGAGTCGCGTACCCGAGCATCGCCTCGGAGATCATCGATCGCTTCAGCGGTAGCCGACCACTCCCGGCCCTGCGGCTCGCGGCCTTGC AGCTGCGCGAAGTAGTCCTTCGGCGACAGGACACCACGCCGGTTCAGGTCGTGGGCCACCAGGTGCAGCGGCTCGTGGTTGTCGACGACGCGGTGATACACCT CGAGGATGCGCTCTCGCTGCACAGGTCCGGCACCAGCCGCCACTCCCCGTCCACGCGCGTAGGCAGGTATCCCCACGGCGGCAGGGATCCTCGGTATTTCCCGGCGCGGATATTGAAATGCGCAGCCGAACGGTTCCGCTCTTTGATCGCTTCTAATTCCATCTGCGCCACCGTTCCCATAAGCGCGATGACGACCGCCGCAAACGGCGTCGTCGTATCGAAGTGCGCTTCGGTCGCGGAGACGACC AGCTTCTTGTGGTCCTCGGCCCAGTGGACCAGCTGTTGCAGATGCCGGATCGATCGGGTCAACCGGTCTACCCGGTACGCCACGATCACGTCGAACGGTTGCTCCTCGAA CGCTAGCCACCGGGCCAGGTTCGGTCTGCGCTTCCGGTCGAACGGATCGACCGCCCCGGAGACGTCCAGATCCTCCGCTACCCCGACGACGTCCCAGCCGCTGGGCGCAGAGCTGCTGGCAAGACTCCAGCTGACGCTCCGGTGAAGTCGTAGCATCGGTGACGCGGGACAGGCGGATGACTACCAGGGCTCTCATCTAGTATTTCTCCTCTTTCTCTAGTATTAAACAAAATTATTTGTAGAGGCTGTTTC GTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCGCAGGTCAAAGGGTATACTGGGATTCCAGTGAACGCAATCAATTTCTGAGAAC TGTCATTCTCGGAAATTGAGGGTTTGTACCGTACACCACTGAGACCGCGGTGGTTGACCAGACAAACCACGAGGGAGACCAGAAACAAAAAAAGGCCCCCCGTTAGGGAGGCCTTCAATAATTGGTTATCATTTGTACAGTTCATCCATACCATGCGTGATGCCCGCTGCGGTTACGAACTCCAGCAGAACCATATGATCGCGTTTCTCGTTCGGATCTTTAGACAGAACGCTTTGCGTGCTCAGATAGTGATTGTCTGGCAGCA GAACAGGACCATCACCGATTGGAGTGTTTTGCTGGTAGTGATCAGCCAGCTGCACGCTGCCATCCTCCACGTTGTGGCGAATTTTAAAATTCG CTTTAATGCCATTTTTTTGTTTATCGCGTGATGTAAACATTGTGGCTGTTAAAATTGTATTCCAGCTTATGGCCCAGGATATTGCCGTCTTCTTTAAAGTCAATGCCTTTCAGCTCAATGCGGTTTACCAGGGTATCGCCTTCAAATTTCACTTCCGCACGCGTTTTTGTACGTGCCGTCATCCTTAAAGGAAATCGTGCGTTCCTGCACATAGCCTTCCGGCATGGCGGACTTGAAGAAGTCATGCTGCTTCATATGGT CCGGATAACGAGCAAAGCACTGAACACCATAAGTCAGCGTCGTTACCAGAGTCGGCCAAGGTACCGGCAGTTTACCAGTAGTACAGATGAACTTCA GCGTCAGTTTACCATTAGTTGCGTCACCTTCACCCTCGCCACGCACGGAAAACTTATGACCGTTGACATCACCATCCAGTTCCACCAGAATAGGGACGACACCAGTGAACAGCTCTTCGCCTTTACGCATCTAGTATTTCTCCTCTTCCTAGTAACTCTTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCCGCAGGTCAAAATATATACTGG GATTCCAGTGAACGCAACAGGATGTGACGAGCGGTGTGGTCAATTTCTGAGAACTGTCATTCTCGGAAATTGAACTGGCCGTCGTTTTACAAC(SEQ ID NO:44)

The sequence of VloxP is:

TCAATTTCCGAGAATGACAGTTCTCAGAAATTGA(SEQ ID NO:45)

The recombinant plasmid pBxb1-attP-VCre was transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha Bxb1-attB by conjugation transformation method, and positive clones were screened on LB plates containing 200 μg/ml kanamycin and 100 μg/ml apramycin, and recombinant bacteria with plasmid integrated into the genome were obtained. The recombinant plasmid pVCre was then transferred into Escherichia coli S17-1, and then transferred into the recombinant bacteria by conjugation transformation method, and 8 clones grown were randomly selected on LB plates containing 250 μg/ml spectinomycin and 100 μg/ml apramycin, and PCR verification was performed using primers tcggcggcggccgggcgtg (SEQ ID NO: 46) and caccgattggagtgttttgc (SEQ ID NO: 47), and all the expected 1547 bp bands were obtained, proving that VCre deleted the vector backbone (Figure 4). Finally, the positive clones were cultured on non-resistant plates to lose the pVCre plasmid, thus obtaining the recombinant bacteria Ralstonia eutropha Bxb1-GFP in which the exogenous gene GFP was integrated into the genome.

Sequence Listing

<110> Shenzhen Blue Crystal Biotechnology Co., Ltd.

<120> A method for integrating exogenous sequences into a genome

<130> DI20-0220-XC37

<160> 47

<170> SIPOSequenceListing 1.0

<210> 1

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> H1-primer 1

<400> 1

acacaggaaa cagctatgac tggtacccgg ccaagtctgc 40

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> H1-primer 2

<400> 2

gatttgattg tctctctgcc 20

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> H2-primer 3

<400> 3

cctgccggcc tggttcaac 19

<210> 4

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> H2-primer 4

<400> 4

gttgtaaaac gacggccagt aaagcctcta ccgctcgc 38

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 5

<400> 5

gtcatagctg tttcctgtgt g 21

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 6

<400> 6

actggccgtc gttttacaac 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 7

<400> 7

ggcagagaga caatcaaatc 20

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 8

<400> 8

gttgaaccag gccggcagg 19

<210> 9

<211> 337

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Clip 01

<400> 9

ggcagagaga caatcaaatc tctagggcgg cggatttgtc ctactcagga gagcgttcac 60

cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg 120

atgcccagga aacagctatg acggttcggc cggcttgtcg acgacggcgg tctccgtcgt 180

caggatcatc cgggcactgg ccgtcgtttt acaaccttgg actcctgttg atagatccag 240

taatgacctc agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt 300

ttattggtga gaatccagcc tgccggcctg gttcaac 347

<210> 10

<211> 50

<212> DNA

<213> Artificial Sequence

<220>

<223> attB sequence corresponding to Bxb1

<400> 10

tcggccggct tgtcgacgac ggcggtctcc gtcgtcagga tcatccgggc 50

<210> 11

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic fragment 02

<400> 11

ggcagagaga caatcaaatc tctagggcgg cggatttgtc ctactcagga gagcgttcac 60

cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg 120

atgcccagga aacagctatg acggtcgcgc ccggggagcc caagggcacg ccctggcaca 180

ctggccgtcg ttttacaacc ttggactcct gttgatagat ccagtaatga cctcagaact 240

ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc 300

agcctgccgg cctggttcaa c 331

<210> 12

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> attB sequence corresponding to PhiC31

<400> 12

cgcgcccggg gagcccaagg gcacgccctg gcac 34

<210> 13

<211> 340

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic fragment 03

<400> 13

ggcagagaga caatcaaatc tctagggcgg cggatttgtc ctactcagga gagcgttcac 60

cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg 120

atgcccagga aacagctatg acggttatgc caacacaatt aacatctcaa tcaaggtaaa 180

tgctttttgc tttttttgac tggccgtcgt tttacaacct tggactcctg ttgatagatc 240

cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt 300

tttttattgg tgagaatcca gcctgccggc ctggttcaac 350

<210> 14

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> attB sequence corresponding to TP901

<400> 14

tatgccaaca caattaacat ctcaatcaag gtaaatgctt tttgcttttt ttg 53

<210> 15

<211> 314

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic Clip 04

<400> 15

ggcagagaga caatcaaatc tctagggcgg cggatttgtc ctactcagga gagcgttcac 60

cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg 120

atgcccagga aacagctatg acggtacgac cttcgcatta cgaatgcgct gcactggccg 180

tcgttttaca accttggact cctgttgata gatccagtaa tgacctcaga actccatctg 240

gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccagcctgc 300

cggcctggtt caac 324

<210> 16

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> attB sequence corresponding to P22

<400> 16

acgaccttcg cattacgaat gcgctgc 27

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 9

<400> 17

cacacaggaa acagctatga c 21

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 10

<400> 18

gttgtaaaac gacggccagt 20

<210> 19

<211> 1844

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic fragment 05

<400> 19

cacacaggaa acagctatga cctggattct caccaataaa aaacgcccgg cggcaaccga 60

gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc 120

caagctacga catcccggtg tgtagccgtt cgaccacgct gccgagcctg agatgctgct 180

cgtactcttg cagatccccg aagtcgatcg tgcgagtcag cccgccgcgg acgtcgaacg 240

tcagccgaac gttcatcgac cgaagccagg tgttctttgc cgcggtgtcc tgctcccgcc 300

accagtcccc gaaccgctgc ccggtctcgc gccactccca gccagacggg cgagcctcta 360

ggccctccag ctcctcttgc cgcgcggcca gcgccgcaat acgggcatcc agtgcttctc 420

gctgcggaga gccggcccgg taggccgggg agccgatcag cgacgtcagg tccaccagct 480

ccgcgttcac ctccgcgagt tcgaccgcgg agtccgagcc ggctacccag actttctcca 540

gacgctccgc gtccccgagc agatccagca cctgctcctc gcagaacgcg tcccactcgg 600

ccatcgccac cgtgccgttc ccgcagtgct tcgggaaccc catcgagcgg cagcggtagc 660

gcgggtgctt acgtcctccc ccggcgaact tgtacgcggg ctccccgcac accgcgcaga 720

acaacacccg cagcagcagc gacggggtag acaccgcggg cttcgcccgg gaggtcttca 780

cgagctcggc gcgcagcgcc tccagctgct cacgggtcag gatcggctca gcccgcacca 840

gcggggctcc gtcgtcgtct cggacggtct taccgttcag agtcgcgtac ccgagcatcg 900

cctcggagat catcgatcgc ttcagcgcgg tagccgacca ctcccggccc tgcggctcgc 960

ggccttgcag ctgcgcgaag tagtccttcg gcgacaggac accacgccgg ttcaggtcgt 1020

gggccaccag gtgcagcggc tcgtggttgt cgacgacgcg gtgatacacc tcgaggatgc 1080

gctctcgctg cacagggtcc ggcaccagcc gccactcccc gtccacgcgc gtaggcaggt 1140

atccccacgg cggcagggat cctcggtatt tcccggcgcg gatattgaaa tgcgcagccg 1200

aacggttccg ctctttgatc gcttctaatt ccatctgcgc caccgttccc ataagcgcga 1260

tgacgaccgc cgcaaacggc gtcgtcgtat cgaagtgcgc ttcggtcgcg gagacgacca 1320

gcttcttgtg gtcctcggcc cagtggacca gctgttgcag atgccggatc gatcgggtca 1380

accggtctac ccggtacgcc acgatcacgt cgaacggttg ctcctcgaac gctagccacc 1440

gggccaggtt cggtctgcgc ttccggtcga acggatcgac cgccccggag acgtccagat 1500

cctccgctac cccgacgacg tcccagccgc gctgggcgca gagctgctgg caagactcca 1560

gctgacgctc cggtgaagtc gtagcatcgg tgacgcggga caggcggatg actaccaggg 1620

ctctcatcta gtatttctcc tctttctcta gtattaaaca aaattatttg tagaggctgt 1680

ttcgtcctca cggactcatc agaccggaaa gcacatccgg tgacagcttg ctcgcaggtc 1740

aaagggtata ctgggattcc agtgaacgca agggtttgta ccgtacacca ctgagaccgc 1800

ggtggttgac cagacaaacc acgaactggc cgtcgtttta caac 1904

<210> 20

<211> 1503

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene sequence of Bxb1 recombinase

<400> 20

atgagagccc tggtagtcat ccgcctgtcc cgcgtcaccg atgctacgac ttcaccggag 60

cgtcagctgg agtcttgcca gcagctctgc gcccagcgcg gctgggacgt cgtcggggta 120

gcggaggatc tggacgtctc cggggcggtc gatccgttcg accggaagcg cagaccgaac 180

ctggcccggt ggctagcgtt cgaggagcaa ccgttcgacg tgatcgtggc gtaccgggta 240

gaccggttga cccgatcgat ccggcatctg caacagctgg tccactgggc cgaggaccac 300

aagaagctgg tcgtctccgc gaccgaagcg cacttcgata cgacgacgcc gtttgcggcg 360

gtcgtcatcg cgctttatggg aacggtggcg cagatggaat tagaagcgat caaagagcgg 420

aaccgttcgg ctgcgcattt caatatccgc gccgggaaat accgaggatc cctgccgccg 480

tggggatacc tgcctacgcg cgtggacggg gagtggcggc tggtgccgga ccctgtgcag 540

cgagagcgca tcctcgaggt gtatcaccgc gtcgtcgaca accacgagcc gctgcacctg 600

gtggcccacg acctgaaccg gcgtggtgtc ctgtcgccga aggactactt cgcgcagctg 660

caaggccgcg agccgcaggg ccggggagtgg tcggctaccg cgctgaagcg atcgatgatc 720

tccgaggcga tgctcgggta cgcgactctg aacggtaaga ccgtccgaga cgacgacgga 780

gccccgctgg tgcgggctga gccgatcctg acccgtgagc agctggaggc gctgcgcgcc 840

gagctcgtga agacctcccg ggcgaagccc gcggtgtcta ccccgtcgct gctgctgcgg 900

gtgttgttct gcgcggtgtg cggggagccc gcgtacaagt tcgccggggg aggacgtaag 960

cacccgcgct accgctgccg ctcgatgggg ttcccgaagc actgcgggaa cggcacggtg 1020

gcgatggccg agtgggacgc gttctgcgag gagcaggtgc tggatctgct cggggacgcg 1080

gagcgtctgg agaaagtctg ggtagccggc tcggactccg cggtcgaact cgcggaggtg 1140

aacgcggagc tggtggacct gacgtcgctg atcggctccc cggcctaccg ggccggctct 1200

ccgcagcgag aagcactgga tgcccgtatt gcggcgctgg ccgcgcggca agaggagctg 1260

gagggcctag aggctcgccc gtctggctgg gagtggcgcg agaccgggca gcggttcggg 1320

gactggtggc gggagcagga caccgcggca aagaacacct ggcttcggtc gatgaacgtt 1380

cggctgacgt tcgacgtccg cggcgggctg actcgcacga tcgacttcgg ggatctgcaa 1440

gagtacgagc agcatctcag gctcggcagc gtggtcgaac ggctacacac cgggatgtcg 1500

tag 1553

<210> 21

<211> 500

<212> PRT

<213> Artificial Sequence

<220>

<223> Bxb1 recombinase

<400> 21

Met Arg Ala Leu Val Val Ile Arg Leu Ser Arg Val Thr Asp Ala Thr

1 5 10 15

Thr Ser Pro Glu Arg Gln Leu Glu Ser Cys Gln Gln Leu Cys Ala Gln

20 25 30

Arg Gly Trp Asp Val Val Gly Val Ala Glu Asp Leu Asp Val Ser Gly

35 40 45

Ala Val Asp Pro Phe Asp Arg Lys Arg Arg Pro Asn Leu Ala Arg Trp

50 55 60

Leu Ala Phe Glu Glu Gln Pro Phe Asp Val Ile Val Ala Tyr Arg Val

65 70 75 80

Asp Arg Leu Thr Arg Ser Ile Arg His Leu Gln Gln Leu Val His Trp

85 90 95

Ala Glu Asp His Lys Lys Leu Val Val Ser Ala Thr Glu Ala His Phe

100 105 110

Asp Thr Thr Thr Pro Phe Ala Ala Val Val Ile Ala Leu Met Gly Thr

115 120 125

Val Ala Gln Met Glu Leu Glu Ala Ile Lys Glu Arg Asn Arg Ser Ala

130 135 140

Ala His Phe Asn Ile Arg Ala Gly Lys Tyr Arg Gly Ser Leu Pro Pro

145 150 155 160

Trp Gly Tyr Leu Pro Thr Arg Val Asp Gly Glu Trp Arg Leu Val Pro

165 170 175

Asp Pro Val Gln Arg Glu Arg Ile Leu Glu Val Tyr His Arg Val Val

180 185 190

Asp Asn His Glu Pro Leu His Leu Val Ala His Asp Leu Asn Arg Arg

195 200 205

Gly Val Leu Ser Pro Lys Asp Tyr Phe Ala Gln Leu Gln Gly Arg Glu

210 215 220

Pro Gln Gly Arg Glu Trp Ser Ala Thr Ala Leu Lys Arg Ser Met Ile

225 230 235 240

Ser Glu Ala Met Leu Gly Tyr Ala Thr Leu Asn Gly Lys Thr Val Arg

245 250 255

Asp Asp Asp Gly Ala Pro Leu Val Arg Ala Glu Pro Ile Leu Thr Arg

260 265 270

Glu Gln Leu Glu Ala Leu Arg Ala Glu Leu Val Lys Thr Ser Arg Ala

275 280 285

Lys Pro Ala Val Ser Thr Pro Ser Leu Leu Leu Arg Val Leu Phe Cys

290 295 300

Ala Val Cys Gly Glu Pro Ala Tyr Lys Phe Ala Gly Gly Gly Arg Lys

305 310 315 320

His Pro Arg Tyr Arg Cys Arg Ser Met Gly Phe Pro Lys His Cys Gly

325 330 335

Asn Gly Thr Val Ala Met Ala Glu Trp Asp Ala Phe Cys Glu Glu Gln

340 345 350

Val Leu Asp Leu Leu Gly Asp Ala Glu Arg Leu Glu Lys Val Trp Val

355 360 365

Ala Gly Ser Asp Ser Ala Val Glu Leu Ala Glu Val Asn Ala Glu Leu

370 375 380

Val Asp Leu Thr Ser Leu Ile Gly Ser Pro Ala Tyr Arg Ala Gly Ser

385 390 395 400

Pro Gln Arg Glu Ala Leu Asp Ala Arg Ile Ala Ala Leu Ala Ala Arg

405 410 415

Gln Glu Glu Leu Glu Gly Leu Glu Ala Arg Pro Ser Gly Trp Glu Trp

420 425 430

Arg Glu Thr Gly Gln Arg Phe Gly Asp Trp Trp Arg Glu Gln Asp Thr

435 440 445

Ala Ala Lys Asn Thr Trp Leu Arg Ser Met Asn Val Arg Leu Thr Phe

450 455 460

Asp Val Arg Gly Gly Leu Thr Arg Thr Ile Asp Phe Gly Asp Leu Gln

465 470 475 480

Glu Tyr Glu Gln His Leu Arg Leu Gly Ser Val Val Glu Arg Leu His

485 490 495

Thr Gly Met Ser

500

<210> 22

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> attP sequence corresponding to Bxb1

<400> 22

tcgtggtttg tctggtcaac caccgcggtc tcagtggtgt acggtacaaa ccc 53

<210> 23

<211> 2145

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic fragment 06

<400> 23

cacacaggaa acagctatga cctggattct caccaataaa aaacgcccgg cggcaaccga 60

gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc 120

caagctacgc cgctacgtct tccgtgccgt cctgggcgtc gtcttcgtcg tcgtcggtcg 180

gcggcttcgc ccacgtgatc gaagcgcgct tctcgatggg cgttccctgc cccctgcccg 240

tagtcgactt cgtgacaacg atcttgtcta cgaagagccc gacgaacacg cgcttgtcgt 300

ctactgacgc gcgcccccac cacgacttag ggccggtcgg gtcagcgtcg gcgtcttcgg 360

ggaaccattg gtcaagggga agcttcgggg cttcggcggc ttcaagttcg gcaagccgct 420

cttccgcccc ttgctgccgg agcgtcagcg ctgcctgttg cttccggaag tgcttcctgc 480

caacgggtcc gtcgtacgcg cctgccgcgc ggtcttcgta cagctcttca agggcgttca 540

gggcgtcggc gcgctccgca acaaggttcg cccgttcgcc gctcttctca ggcgcctcag 600

tgagcttgcc gaagcgtcgg gcggcttccc acagaagcgc caacgtctct tcgtcgcctt 660

cggcgtgcct gatcttgttg aagatgcgtt ccgcaacgaa cttgtcgagt gccgccatgc 720

tgacgttgca cgtgccttcg tgctgcccag gtgcggacgg gtcgaccacc ttccggcgac 780

ggcagcggta agagtccttg atcgattctt ccccgcgctt cgaagtcatg acggcgccac 840

actcgcagta cagcttgtcc atggcggaca gaatggcttg cccccgggaa agccccttgc 900

cgcgccccct gccgtccaac cacgcctgaa gctcatacca ctcagcgggc tcgatgatcg 960

gtccgcaatc aagctcgacc ggccggagcg tgatcgggtc gcgctgaatg cggtaaccct 1020

caatcttcgt ggtcggcgtg ccgtccggct tcttcttgta gatcacctca gcggcgaagc 1080

ccgcaatacg cgggtcccga aggattcgca taacggttgc cgggtcccag gcgcttgaag 1140

cggtcttctt cccaatcgtc tcgccccggg tcggcacggc gtcagcgtcc atgcgcttac 1200

aaagccccgt gatgctgccc gggtgaatgg cggcttgact gcccggcttg aagggaaggt 1260

gtttgtgcgt cttgatctca cgccaccacc accggattac gtcgggctcg aactcgaagg 1320

gtccggtaag gggagtggtc gagtgcgcaa gcttgttgat gacgacattg accattcggc 1380

cgttgcgcgt gatctccttc gtctccgaaa caagctcgaa gccgtaaggc gccttcccgc 1440

cgacgtaccc gcccaattcg cgctgaaggt tcttcgtgtc gagaatcttc gccgacttca 1500

gcgaagattc tttgtgcgac gcgtcgagcc gcataatcag gtgaatcagg tccatgacgt 1560

ttccctgccg gaagacgcct tcctgagtgg aaacaatcgt cacgcccagg gcgagcaatt 1620

ccgagacaat cggaatcgcg tccatgacct tcaggcgcga gaagcgcgac acgtcataga 1680

caatgatcat gttgagccgc ccggcgcggc attcgttcag gatgcgttcg aactccgggc 1740

gctccgccgt cccgaacgcc gacgtgcccg gcgcttcgct gaaatgcccg acgaacctga 1800

accggccccc gtcgcgctcg acttcgcgct gaaggtcggc cgccttgtct tcgttggcgc 1860

tacgctgtgt cgctgggctt gctgcgctcg aattctcgcg ctcgcgcgac tgacggtcgt 1920

aagcacccgc gtacgtgtcc atctagtatt tctcctcttt ctctagtatt aaacaaaatt 1980

atttgtagag gctgtttcgt cctcacggac tcatcagacc ggaaagcaca tccggtgaca 2040

gcttgctcgc aggtcaaagg gtatactggg attccagtga acgcaacccc aactggggta 2100

acctttgagt tctctcagtt gggggactgg ccgtcgtttt acaac 2215

<210> 24

<211> 1818

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene sequence of PhiC31 recombinase

<400> 24

atggacacgt acgcgggtgc ttacgaccgt cagtcgcgcg agcgcgagaa ttcgagcgca 60

gcaagcccag cgacacagcg tagcgccaac gaagacaagg cggccgacct tcagcgcgaa 120

gtcgagcgcg acgggggccg gttcaggttc gtcgggcatt tcagcgaagc gccgggcacg 180

tcggcgttcg ggacggcgga gcgcccggag ttcgaacgca tcctgaacga atgccgcgcc 240

gggcggctca acatgatcat tgtctatgac gtgtcgcgct tctcgcgcct gaaggtcatg 300

gacgcgattc cgattgtctc ggaattgctc gccctgggcg tgacgattgt ttccactcag 360

gaaggcgtct tccggcaggg aaacgtcatg gacctgattc acctgattat gcggctcgac 420

gcgtcgcaca aagaatcttc gctgaagtcg gcgaagattc tcgacacgaa gaaccttcag 480

cgcgaattgg gcgggtacgt cggcgggaag gcgccttacg gcttcgagct tgtttcggag 540

acgaaggaga tcacgcgcaa cggccgaatg gtcaatgtcg tcatcaacaa gcttgcgcac 600

tcgaccactc cccttaccgg acccttcgag ttcgagcccg acgtaatccg gtggtggtgg 660

cgtgagatca agacgcacaa acaccttccc ttcaagccgg gcagtcaagc cgccattcac 720

ccgggcagca tcacggggct ttgtaagcgc atggacgctg acgccgtgcc gacccggggc 780

gagacgattg ggaagaagac cgcttcaagc gcctgggacc cggcaaccgt tatgcgaatc 840

cttcgggacc cgcgtattgc gggcttcgcc gctgaggtga tctacaagaa gaagccggac 900

ggcacgccga ccacgaagat tgagggttac cgcattcagc gcgacccgat cacgctccgg 960

ccggtcgagc ttgattgcgg accgatcatc gagcccgctg agtggtatga gcttcaggcg 1020

tggttggacg gcaggggggcg cggcaagggg ctttcccggg ggcaagccat tctgtccgcc 1080

atggacaagc tgtactgcga gtgtggcgcc gtcatgactt cgaagcgcgg ggaagaatcg 1140

atcaaggact cttaccgctg ccgtcgccgg aaggtggtcg acccgtccgc acctgggcag 1200

cacgaaggca cgtgcaacgt cagcatggcg gcactcgaca agttcgttgc ggaacgcatc 1260

ttcaacaaga tcaggcacgc cgaaggcgac gaagagacgt tggcgcttct gtgggaagcc 1320

gcccgacgct tcggcaagct cactgaggcg cctgagaaga gcggcgaacg ggcgaacctt 1380

gttgcggagc gcgccgacgc cctgaacgcc cttgaagagc tgtacgaaga ccgcgcggca 1440

ggcgcgtacg acggacccgt tggcaggaag cacttccgga agcaacaggc agcgctgacg 1500

ctccggcagc aaggggcgga agagcggctt gccgaacttg aagccgccga agccccgaag 1560

cttccccttg accaatggtt ccccgaagac gccgacgctg acccgaccgg ccctaagtcg 1620

tggtgggggc gcgcgtcagt agacgacaag cgcgtgttcg tcgggctctt cgtagacaag 1680

atcgttgtca cgaagtcgac tacgggcagg gggcagggaa cgcccatcga gaagcgcgct 1740

tcgatcacgt gggcgaagcc gccgaccgac gacgacgaag acgacgccca ggacggcacg 1800

gaagacgtag cggcgtag 1878

<210> 25

<211> 605

<212> PRT

<213> Artificial Sequence

<220>

<223> PhiC31 recombinase

<400> 25

Met Asp Thr Tyr Ala Gly Ala Tyr Asp Arg Gln Ser Arg Glu Arg Glu

1 5 10 15

Asn Ser Ser Ala Ala Ser Pro Ala Thr Gln Arg Ser Ala Asn Glu Asp

20 25 30

Lys Ala Ala Asp Leu Gln Arg Glu Val Glu Arg Asp Gly Gly Arg Phe

35 40 45

Arg Phe Val Gly His Phe Ser Glu Ala Pro Gly Thr Ser Ala Phe Gly

50 55 60

Thr Ala Glu Arg Pro Glu Phe Glu Arg Ile Leu Asn Glu Cys Arg Ala

65 70 75 80

Gly Arg Leu Asn Met Ile Ile Val Tyr Asp Val Ser Arg Phe Ser Arg

85 90 95

Leu Lys Val Met Asp Ala Ile Pro Ile Val Ser Glu Leu Leu Ala Leu

100 105 110

Gly Val Thr Ile Val Ser Thr Gln Glu Gly Val Phe Arg Gln Gly Asn

115 120 125

Val Met Asp Leu Ile His Leu Ile Met Arg Leu Asp Ala Ser His Lys

130 135 140

Glu Ser Ser Leu Lys Ser Ala Lys Ile Leu Asp Thr Lys Asn Leu Gln

145 150 155 160

Arg Glu Leu Gly Gly Tyr Val Gly Gly Lys Ala Pro Tyr Gly Phe Glu

165 170 175

Leu Val Ser Glu Thr Lys Glu Ile Thr Arg Asn Gly Arg Met Val Asn

180 185 190

Val Val Ile Asn Lys Leu Ala His Ser Thr Thr Pro Leu Thr Gly Pro

195 200 205

Phe Glu Phe Glu Pro Asp Val Ile Arg Trp Trp Trp Arg Glu Ile Lys

210 215 220

Thr His Lys His Leu Pro Phe Lys Pro Gly Ser Gln Ala Ala Ile His

225 230 235 240

Pro Gly Ser Ile Thr Gly Leu Cys Lys Arg Met Asp Ala Asp Ala Val

245 250 255

Pro Thr Arg Gly Glu Thr Ile Gly Lys Lys Thr Ala Ser Ser Ala Trp

260 265 270

Asp Pro Ala Thr Val Met Arg Ile Leu Arg Asp Pro Arg Ile Ala Gly

275 280 285

Phe Ala Ala Glu Val Ile Tyr Lys Lys Lys Pro Asp Gly Thr Pro Thr

290 295 300

Thr Lys Ile Glu Gly Tyr Arg Ile Gln Arg Asp Pro Ile Thr Leu Arg

305 310 315 320

Pro Val Glu Leu Asp Cys Gly Pro Ile Ile Glu Pro Ala Glu Trp Tyr

325 330 335

Glu Leu Gln Ala Trp Leu Asp Gly Arg Gly Arg Gly Lys Gly Leu Ser

340 345 350

Arg Gly Gln Ala Ile Leu Ser Ala Met Asp Lys Leu Tyr Cys Glu Cys

355 360 365

Gly Ala Val Met Thr Ser Lys Arg Gly Glu Glu Ser Ile Lys Asp Ser

370 375 380

Tyr Arg Cys Arg Arg Arg Lys Val Val Asp Pro Ser Ala Pro Gly Gln

385 390 395 400

His Glu Gly Thr Cys Asn Val Ser Met Ala Ala Leu Asp Lys Phe Val

405 410 415

Ala Glu Arg Ile Phe Asn Lys Ile Arg His Ala Glu Gly Asp Glu Glu

420 425 430

Thr Leu Ala Leu Leu Trp Glu Ala Ala Arg Arg Phe Gly Lys Leu Thr

435 440 445

Glu Ala Pro Glu Lys Ser Gly Glu Arg Ala Asn Leu Val Ala Glu Arg

450 455 460

Ala Asp Ala Leu Asn Ala Leu Glu Glu Leu Tyr Glu Asp Arg Ala Ala

465 470 475 480

Gly Ala Tyr Asp Gly Pro Val Gly Arg Lys His Phe Arg Lys Gln Gln

485 490 495

Ala Ala Leu Thr Leu Arg Gln Gln Gly Ala Glu Glu Arg Leu Ala Glu

500 505 510

Leu Glu Ala Ala Glu Ala Pro Lys Leu Pro Leu Asp Gln Trp Phe Pro

515 520 525

Glu Asp Ala Asp Ala Asp Pro Thr Gly Pro Lys Ser Trp Trp Gly Arg

530 535 540

Ala Ser Val Asp Asp Lys Arg Val Phe Val Gly Leu Phe Val Asp Lys

545 550 555 560

Ile Val Val Thr Lys Ser Thr Thr Gly Arg Gly Gln Gly Thr Pro Ile

565 570 575

Glu Lys Arg Ala Ser Ile Thr Trp Ala Lys Pro Pro Thr Asp Asp Asp

580 585 590

Glu Asp Asp Ala Gln Asp Gly Thr Glu Asp Val Ala Ala

595 600 605

<210> 26

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> attP sequence corresponding to PhiC31

<400> 26

cccccaactg agagaactca aaggttaccc cagttgggg 39

<210> 27

<211> 1865

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic fragment 07

<400> 27

cacacaggaa acagctatga cctggattct caccaataaa aaacgcccgg cggcaaccga 60

gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc 120

caagttaagc agccagagcg tagttttcgt ccttagcagc accggtagcg agttggaatt 180

taaatatgat atctacatta tcagcagtaa catcaacctt tgatacaagg ttgttgacga 240

ttttcttttt attatcatat gatagttcat taatcggaat tgagcccaac tgagttttaa 300

ctaactcaaa aacatcagta gagtcattaa atttattttc gctaatctta gctttaagca 360

gctttttctc agcctgaagg gaatcagtac gatctttcaa ctcatccata gtgataaaat 420

catttaggta caaatcagag ttcttttgta tttttttatc gatctgtgaa atttgctttt 480

taaatgacga agtatcaaga ataggttggt tgttgccatt gataattttc aataaggagt 540

cattattttc ttgaaatcca atcaggttgt caataacagt attttctaaa ttaacttaaat 600

cataagttcc tgaatcacac tttttattgt cattatatac tgtaattcct tttgtttttc 660

gaggaaatct atttgcacag tgatatttca tagtgcggct tccatctttt cttttgtggc 720

caagaacaat ttttaaaggt gctccacagt aaccgcacct tgccatccct gacagcatat 780

atttagcttg gaaaggtcta gggttgttat ttctttcata agtctgctgt tgtctttctt 840

ctagctctttttgaactttt aaataagtct cataagggat aattggtttg tgcatacctt 900

caaataggct gtccttaaat ttgatataac cacagtaaac tggattatca agtgtttgtc 960

ttagggtacg ataagaccac ggtatatctt taccgatgtg tccagattca ttgagtttat 1020

ctcttaattt tgtaagtgat attcctgata aataatcagt gaatatttgt tcaactattg 1080

tagcttgtaa aggaacaatt tctaatatac ctgtctttct gttgtggtaa tacccaaaag 1140

ctgtcttagt ccacatcata gacttaccag atttcgctcg ccctagttta cccatagtca 1200

tgcgttcttt tatattctct ctttcaaact cattaattgc agaaagaata gtgagaaaca 1260

agctacccat agcagaagaa gtatcaatac tttcattaag cgagataaag tctattttat 1320

tttttgtgaa cacatcctta acaagataaa gagtatctct tacactacgt gaaaggcggt 1380

ctagcttata tacaagaact gtatcaaaag ctttattctc gatatcgttg attaatcttt 1440

gcattgctgg gcgttcaagt ttggcccctg aaaaaccagc atcagtataa gtatcagata 1500

cttgccaccc cattgcttca gcatattttg ttaaacggtc aatttgctca tcaattgaga 1560

agccttcctc tgcttggtta gtagtggata ctcgtgtata gattgctact ttcttagtgc 1620

cggcctggtg gtgatggtga tgatgtttca tctagtattt ctcctctttc tctagtatta 1680

aacaaaatta tttgtagagg ctgtttcgtc ctcacggact catcagaccg gaaagcacat 1740

ccggtgacag cttgctcgca ggtcaaaggg tatactggga ttccagtgaa cgcaaaaaag 1800

gagtttttta gttaccttaa ttgaaataaa cgaaataaaa actcgactgg ccgtcgtttt 1860

acaac 1927

<210> 28

<211> 1527

<212> DNA

<213> Artificial Sequence

<220>

<223> TP901 recombinase gene sequence

<400> 28

atgaaacatc atcaccatca ccaccaggcc ggcactaaga aagtagcaat ctatacacga 60

gtatccacta ctaaccaagc agaggaaggc ttctcaattg atgagcaaat tgaccgttta 120

acaaaatatg ctgaagcaat ggggtggcaa gtatctgata cttatactga tgctggtttt 180

tcaggggcca aacttgaacg cccagcaatg caaagattaa tcaacgatat cgagaataaa 240

gcttttgata cagttcttgt atataagcta gaccgccttt cacgtagtgt aagagatact 300

ctttatcttg ttaaggatgt gttcacaaaa aataaaatag actttatctc gcttaatgaa 360

agtattgata cttcttctgc tatgggtagc ttgtttctca ctattctttc tgcaattaat 420

gagtttgaaa gagagaatat aaaagaacgc atgactatgg gtaaactagg gcgagcgaaa 480

tctggtaagt ctatgatgtg gactaagaca gcttttgggt attaccacaa cagaaagaca 540

ggtatattag aaattgttcc tttacaagct acaatagttg aacaaatatt cactgattat 600

ttatcaggaa tatcacttac aaaattaaga gataaactca atgaatctgg acacatcggt 660

aaagatatac cgtggtctta tcgtacccta agacaaacac ttgataatcc agtttatactgt 720

ggttatatca aatttaagga cagcctattt gaaggtatgc acaaaccaat tatccccttat 780

gagacttatt taaaagttca aaaagagcta gaagaaagac aacagcagac ttatgaaaga 840

aataacaacc ctagaccttt ccaagctaaa tatatgctgt cagggatggc aaggtgcggt 900

tactgtggag cacctttaaa aattgttctt ggccacaaaa gaaaagatgg aagccgcact 960

atgaaatatc actgtgcaaa tagatttcct cgaaaaacaa aaggaattac agtatataat 1020

gacaataaaa agtgtgattc aggaacttat gatttaagta atttagaaaa tactgttatt 1080

gacaacctga ttggatttca agaaaataat gactccttat tgaaaattat caatggcaac 1140

aaccaaccta ttcttgatac ttcgtcattt aaaaagcaaa tttcacagat cgataaaaaa 1200

atacaaaaga actctgattt gtacctaaat gattttatca ctatggatga gttgaaagat 1260

cgtactgatt cccttcaggc tgagaaaaag ctgcttaaag ctaagattag cgaaaataaa 1320

tttaatgact ctactgatgt ttttgagtta gttaaaactc agttgggctc aattccgatt 1380

aatgaactat catatgataa taaaaagaaa atcgtcaaca accttgtatc aaaggttgat 1440

gttactgctg ataatgtaga tatcatattt aaattccaac tcgctaccgg tgctgctaag 1500

gacgaaaact acgctctggc tgcttaa 1577

<210> 29

<211> 508

<212> PRT

<213> Artificial Sequence

<220>

<223> TP901 recombinase

<400> 29

Met Lys His His His His His Gln Ala Gly Thr Lys Lys Val Ala

1 5 10 15

Ile Tyr Thr Arg Val Ser Thr Thr Asn Gln Ala Glu Glu Gly Phe Ser

20 25 30

Ile Asp Glu Gln Ile Asp Arg Leu Thr Lys Tyr Ala Glu Ala Met Gly

35 40 45

Trp Gln Val Ser Asp Thr Tyr Thr Asp Ala Gly Phe Ser Gly Ala Lys

50 55 60

Leu Glu Arg Pro Ala Met Gln Arg Leu Ile Asn Asp Ile Glu Asn Lys

65 70 75 80

Ala Phe Asp Thr Val Leu Val Tyr Lys Leu Asp Arg Leu Ser Arg Ser

85 90 95

Val Arg Asp Thr Leu Tyr Leu Val Lys Asp Val Phe Thr Lys Asn Lys

100 105 110

Ile Asp Phe Ile Ser Leu Asn Glu Ser Ile Asp Thr Ser Ser Ala Met

115 120 125

Gly Ser Leu Phe Leu Thr Ile Leu Ser Ala Ile Asn Glu Phe Glu Arg

130 135 140

Glu Asn Ile Lys Glu Arg Met Thr Met Gly Lys Leu Gly Arg Ala Lys

145 150 155 160

Ser Gly Lys Ser Met Met Trp Thr Lys Thr Ala Phe Gly Tyr Tyr His

165 170 175

Asn Arg Lys Thr Gly Ile Leu Glu Ile Val Pro Leu Gln Ala Thr Ile

180 185 190

Val Glu Gln Ile Phe Thr Asp Tyr Leu Ser Gly Ile Ser Leu Thr Lys

195 200 205

Leu Arg Asp Lys Leu Asn Glu Ser Gly His Ile Gly Lys Asp Ile Pro

210 215 220

Trp Ser Tyr Arg Thr Leu Arg Gln Thr Leu Asp Asn Pro Val Tyr Cys

225 230 235 240

Gly Tyr Ile Lys Phe Lys Asp Ser Leu Phe Glu Gly Met His Lys Pro

245 250 255

Ile Ile Pro Tyr Glu Thr Tyr Leu Lys Val Gln Lys Glu Leu Glu Glu

260 265 270

Arg Gln Gln Gln Thr Tyr Glu Arg Asn Asn Asn Pro Arg Pro Phe Gln

275 280 285

Ala Lys Tyr Met Leu Ser Gly Met Ala Arg Cys Gly Tyr Cys Gly Ala

290 295 300

Pro Leu Lys Ile Val Leu Gly His Lys Arg Lys Asp Gly Ser Arg Thr

305 310 315 320

Met Lys Tyr His Cys Ala Asn Arg Phe Pro Arg Lys Thr Lys Gly Ile

325 330 335

Thr Val Tyr Asn Asp Asn Lys Lys Cys Asp Ser Gly Thr Tyr Asp Leu

340 345 350

Ser Asn Leu Glu Asn Thr Val Ile Asp Asn Leu Ile Gly Phe Gln Glu

355 360 365

Asn Asn Asp Ser Leu Leu Lys Ile Ile Asn Gly Asn Asn Gln Pro Ile

370 375 380

Leu Asp Thr Ser Ser Phe Lys Lys Gln Ile Ser Gln Ile Asp Lys Lys

385 390 395 400

Ile Gln Lys Asn Ser Asp Leu Tyr Leu Asn Asp Phe Ile Thr Met Asp

405 410 415

Glu Leu Lys Asp Arg Thr Asp Ser Leu Gln Ala Glu Lys Lys Leu Leu

420 425 430

Lys Ala Lys Ile Ser Glu Asn Lys Phe Asn Asp Ser Thr Asp Val Phe

435 440 445

Glu Leu Val Lys Thr Gln Leu Gly Ser Ile Pro Ile Asn Glu Leu Ser

450 455 460

Tyr Asp Asn Lys Lys Lys Ile Val Asn Asn Leu Val Ser Lys Val Asp

465 470 475 480

Val Thr Ala Asp Asn Val Asp Ile Ile Phe Lys Phe Gln Leu Ala Thr

485 490 495

Gly Ala Ala Lys Asp Glu Asn Tyr Ala Leu Ala Ala

500 505

<210> 30

<211> 50

<212> DNA

<213> Artificial Sequence

<220>

<223> attP sequence corresponding to TP901

<400> 30

cgagttttta tttcgtttat ttcaattaag gtaactaaaa aactcctttt 50

<210> 31

<211> 1712

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic fragment 08

<400> 31

cacacaggaa acagctatga cctggattct caccaataaa aaacgcccgg cggcaaccga 60

gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc 120

caagctacgt attattcgtg ccttccttat ttttactgtg ggacatattt gggacagaag 180

taccaaaaat cgagtcaatt tgtcgagcat gttcagtcag gtgatttggt gccagatgag 240

catatcggcg aaccatttcg atagactccc agccacccat ttcctgcaat accgaaatcg 300

gaacgccagc ctgaactaac caacttgccc acgtgtgcct caggtcatga aaacggaagt 360

cttcaatgcc cgctcgtttt aatgctgccc tccatgcagt attagcgtca tagcgcatct 420

tcctcactac aggtgattta gttccgtctg gtttggtgct gctttccttg tagacgaaca 480

cccatttgtg atgattgccg atttgctttt tcagcacccg gcaagcggta tcattcagcg 540

ccactccaat ggcatgatta gacttgcttt gttccgggtg tatccatgcc acctttcgtt 600

gcatgtctat ctgctgccac tccagattga taatgttaga ccgccttaag ccagtagaaa 660

gcgcaaactc tacgactgac tttagcggtt cctggcattc atcaatcaac ctttttgcct 720

cgtgaggctc aagccagcgg atacgcttat ttttcggctg aggaactttg atgatcggag 780

ccttatccag catcttccat tcgcgttcag cagcccggag gagtgcctta atgaatgaaa 840

ggtgagttgc ttttgtagct actgctgccg gcttaggctt gaataccgga ggctgcttcc 900

cattcttcct gcatgcttca tccattaact tccagttttc ctcatgccgc cgattagtta 960

tcttctggat ggcggagtaa atcttcgtct cggtaatatc cttcaactgc attcctgcaa 1020

aatgctggag ccagaatcct atccgactct tgtcatcatc cagcgacttc ttatgcgcct 1080

tctcctctaa ccacctgaca caggccccct caaaagtcat gtcaggcgtc tctcctaatt 1140

tacttaccct ccatgcttct gccttcagct tgtcatgaag ctctgtggcc tgccttttgt 1200

cctttgtccc aagagactgc ttaaatcttt tgccgttcgg caatgtgaaa ctggcgtacc 1260

aggtttcacc tctgcggaat agtgacatct agtatttctc ctctttctct agtattaaac 1320

aaaattattt gtagaggctg tttcgtcctc acggactcat cagaccggaa agcacatccg 1380

gtgacagctt gctcgcaggt caaagggtat actgggattc cagtgaacgc aactaagtgg 1440

tttgggacaa aaatgggaca tacaaatctt tgcatcggtt tgcaaggctt tgcatgtctt 1500

tcgaagatgg gacgtgtgag cgcaggtatg acgtggtatg ttgttgactt aaaaggtagt 1560

tcttataatt cgtaatgcga aggtcgtagg ttcgactcct attatcggca ccagttaaat 1620

caaatactta cgtattattc gtgccttcct tatttttatact gtgggacata tttggggacag 1680

aagtaccaaa aaactggccg tcgttttaca ac 1768

<210> 32

<211> 1164

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene sequence of P22 recombinase

<400> 32

atgtcactat tccgcagagg tgaaacctgg tacgccagtt tcacattgcc gaacggcaaa 60

agatttaagc agtctcttgg gacaaaggac aaaaggcagg ccacagagct tcatgacaag 120

ctgaaggcag aagcatggag ggtaagtaaa ttaggagaga cgcctgacat gacttttgag 180

ggggcctgtg tcaggtggtt agaggagaag gcgcataaga agtcgctgga tgatgacaag 240

agtcggatag gattctggct ccagcatttt gcaggaatgc agttgaagga tattaccgag 300

acgaagattt actccgccat ccagaagata actaatcggc ggcatgagga aaactggaag 360

ttaatggatg aagcatgcag gaagaatggg aagcagcctc cggtattcaa gcctaagccg 420

gcagcagtag ctacaaaagc aactcacctt tcattcatta aggcactcct ccgggctgct 480

gaacgcgaat ggaagatgct ggataaggct ccgatcatca aagttcctca gccgaaaaat 540

aagcgtatcc gctggcttga gcctcacgag gcaaaaaggt tgattgatga atgccaggaa 600

ccgctaaagt cagtcgtaga gtttgcgctt tctactggct taaggcggtc taacattatc 660

aatctggagt ggcagcagat agacatgcaa cgaaaggtgg catggataca cccggaacaa 720

agcaagtcta atcatgccat tggagtggcg ctgaatgata ccgcttgccg ggtgctgaaa 780

aagcaaatcg gcaatcatca caaatgggtg ttcgtctaca aggaaagcag caccaaacca 840

gacggaacta aatcacctgt agtgaggaag atgcgctatg acgctaatac tgcatggagg 900

gcagcattaa aacgagcggg cattgaagac ttccgttttc atgacctgag gcacacgtgg 960

gcaagttggt tagttcaggc tggcgttccg atttcggtat tgcaggaaat gggtggctgg 1020

gagtctatcg aaatggttcg ccgatatgct catctggcac caaatcacct gactgaacat 1080

gctcgacaaa ttgactcgat ttttggtact tctgtcccaa atatgtccca cagtaaaaat 1140

aaggaaggca cgaataatac gtag 1202

<210> 33

<211> 387

<212> PRT

<213> Artificial Sequence

<220>

<223> P22 recombinase

<400> 33

Met Ser Leu Phe Arg Arg Gly Glu Thr Trp Tyr Ala Ser Phe Thr Leu

1 5 10 15

Pro Asn Gly Lys Arg Phe Lys Gln Ser Leu Gly Thr Lys Asp Lys Arg

20 25 30

Gln Ala Thr Glu Leu His Asp Lys Leu Lys Ala Glu Ala Trp Arg Val

35 40 45

Ser Lys Leu Gly Glu Thr Pro Asp Met Thr Phe Glu Gly Ala Cys Val

50 55 60

Arg Trp Leu Glu Glu Lys Ala His Lys Lys Ser Leu Asp Asp Asp Lys

65 70 75 80

Ser Arg Ile Gly Phe Trp Leu Gln His Phe Ala Gly Met Gln Leu Lys

85 90 95

Asp Ile Thr Glu Thr Lys Ile Tyr Ser Ala Ile Gln Lys Ile Thr Asn

100 105 110

Arg Arg His Glu Glu Asn Trp Lys Leu Met Asp Glu Ala Cys Arg Lys

115 120 125

Asn Gly Lys Gln Pro Pro Val Phe Lys Pro Lys Pro Ala Ala Val Ala

130 135 140

Thr Lys Ala Thr His Leu Ser Phe Ile Lys Ala Leu Leu Arg Ala Ala

145 150 155 160

Glu Arg Glu Trp Lys Met Leu Asp Lys Ala Pro Ile Ile Lys Val Pro

165 170 175

Gln Pro Lys Asn Lys Arg Ile Arg Trp Leu Glu Pro His Glu Ala Lys

180 185 190

Arg Leu Ile Asp Glu Cys Gln Glu Pro Leu Lys Ser Val Val Glu Phe

195 200 205

Ala Leu Ser Thr Gly Leu Arg Arg Ser Asn Ile Ile Asn Leu Glu Trp

210 215 220

Gln Gln Ile Asp Met Gln Arg Lys Val Ala Trp Ile His Pro Glu Gln

225 230 235 240

Ser Lys Ser Asn His Ala Ile Gly Val Ala Leu Asn Asp Thr Ala Cys

245 250 255

Arg Val Leu Lys Lys Gln Ile Gly Asn His His Lys Trp Val Phe Val

260 265 270

Tyr Lys Glu Ser Ser Thr Lys Pro Asp Gly Thr Lys Ser Pro Val Val

275 280 285

Arg Lys Met Arg Tyr Asp Ala Asn Thr Ala Trp Arg Ala Ala Leu Lys

290 295 300

Arg Ala Gly Ile Glu Asp Phe Arg Phe His Asp Leu Arg His Thr Trp

305 310 315 320

Ala Ser Trp Leu Val Gln Ala Gly Val Pro Ile Ser Val Leu Gln Glu

325 330 335

Met Gly Gly Trp Glu Ser Ile Glu Met Val Arg Arg Tyr Ala His Leu

340 345 350

Ala Pro Asn His Leu Thr Glu His Ala Arg Gln Ile Asp Ser Ile Phe

355 360 365

Gly Thr Ser Val Pro Asn Met Ser His Ser Lys Asn Lys Glu Gly Thr

370 375 380

Asn Asn Thr

385

<210> 34

<211> 260

<212> DNA

<213> Artificial Sequence

<220>

<223> attP sequence corresponding to P22

<400> 34

tttttggtac ttctgtccca aatatgtccc acagtaaaaa taaggaaggc acgaataata 60

cgtaagtatt tgatttaact ggtgccgata ataggagtcg aacctacgac cttcgcatta 120

cgaattataa gaactacctt ttaagtcaac aacataccac gtcatacctg cgctcacacg 180

tcccatcttc gaaagacatg caaagccttg caaaccgatg caaagatttg tatgtcccat 240

ttttgtccca aaccacttag 268

<210> 35

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 35

gcgcatggcg tctccatg 18

<210> 36

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 36

gtggaccagc tgttgcag 18

<210> 37

<211> 17

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 11

<400> 37

ctaccggcgc ggcagcg 17

<210> 38

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 12

<400> 38

gcggccaccg gctggctc 18

<210> 39

<211> 17

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 13

<400> 39

cgctgccgcg ccggtag 17

<210> 40

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 14

<400> 40

gagccagccg gtggccgc 18

<210> 41

<211> 5555

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic fragment 09

<400> 41

gagccagccg gtggccgcct acatggctct gctgtagttc acccttggcg tccaaccagc 60

ggcaccagcg gcgcctgaga ggggcgcgcc cagctgtcta gggcggcgga tttgtcctac 120

tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag 180

cctttcgttt tatttgatgc ctttaattaa agcggataac aatttcacac aggacaactg 240

agaccggaat tggtctcaac gtacgtctca ttttcgccag atatcgacgt cttaagaccc 300

actttcacat ttaagttgtt tttctaatcc gcatatgatc aattcaaggc cgaataagaa 360

ggctggctct gcaccttggt gatcaaataa ttcgatagct tgtcgtaata atggcggcat 420

actatcagta gtaggtgttt ccctttcttc tttagcgact tgatgctctt gatcttccaa 480

tacgcaacct aaagtaaaat gccccacagc gctgagtgca tataatgcat tctctagtga 540

aaaaccttgt tggcataaaa aggctaattg attttcgaga gtttcatact gtttttctgt 600

aggccgtgta cctaaatgta cttttgctcc atcgcgatga cttagtaaag cacatctaaa 660

acttttagcg ttattacgta aaaaatcttg ccagctttcc ccttctaaag ggcaaaagtg 720

agtatggtgc ctatctaaca tctcaatggc taaggcgtcg agcaaagccc gcttattttt 780

tacatgccaa tacaatgtag gctgctctac acctagcttc tgggcgagtt tacgggttgt 840

taaaccttcg attccgacct cattaagcag ctctaatgcg ctgttaatca ctttactttt 900

atctaatcta gacatcatta attcctaatt tttgttgaca ctctatcgtt gatagagtta 960

ttttaccact ccctatcagt gatagagaaa agaattcaag ctgtcaccgg atgtgctttc 1020

cggtctgatg agtccgtgag gacgaaacag cctctacaaa taattttgtt taatactaga 1080

gaaagaggag aaatactaga tgatcgagaa ccagctgagc ctgctgggtg atttcagcgg 1140

cgtgcgtccg gacgatgtta agaccgcgat ccaggcggcg caaaagaaag gtattaacgt 1200

tgcggagaac gaacaattca aagcggcgtt tgagcacctg ctgaacgagt tcaagaaacg 1260

tgaggaacgt tacagcccga acaccctgcg tcgtctggaa agcgcgtgga cctgctttgt 1320

ggattggtgc ctggcgaacc atcgtcacag cctgccggcg accccggaca ccgttgaggc 1380

gttctttatc gaacgtgcgg aggaactgca ccgtaacacc ctgagcgtgt accgttgggc 1440

gattagccgt gttcatcgtg ttgcgggttg cccggacccg tgcctggata tctatgtgga 1500

ggatcgtctg aaggcgattg cgcgtaagaa agtgcgtgag ggcgaagcgg ttaaacaggc 1560

gagcccgttt aacgaacaac acctgctgaa gctgaccagc ctgtggtacc gtagcgacaa 1620

actgctgctg cgtcgtaacc tggcgctgct ggcggtggcg tatgagagca tgctgcgtgc 1680

gagcgaactg gcgaacatcc gtgttagcga catggagctg gcgggtgatg gcaccgcgat 1740

tctgaccatc ccgattacca agaccaacca cagcggcgag ccggacacct gcattctgag 1800

ccaggatgtg gttagcctgc tgatggacta caccgaagcg ggcaagctgg acatgagcag 1860

cgatggtttc ctgtttgtgg gcgttagcaa acacaacacc tgcatcaagc cgaagaaaga 1920

taaacagacc ggtgaagttc tgcacaagcc gattaccacc aaaaccgtgg agggcgtttt 1980

ctatagcgcg tgggaaaccc tggatctggg tcgtcaaggc gtgaagccgt ttaccgcgca 2040

cagcgcgcgt gttggtgcgg cgcaggacct gctgaagaaa ggctacaaca ccctgcaaat 2100

ccagcaaagc ggtcgttgga gcagcggcgc gatggttgcg cgttatggtc gtgcgatcct 2160

ggcgcgtgac ggcgcgatgg cgcacagccg tgtgaaaacc cgtagcgcgc cgatgcaatg 2220

gggcaaggac gagaaagatt aatgataagc caggcatcaa ataaaacgaa aggctcagtc 2280

gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca 2340

cactggctca ccttcgggtg ggcctttctg cgtttatata ctagagctgc taacaaagcc 2400

cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg 2460

gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattacta 2520

gaggtcatgc ttgccatctg ttttcttgca agattactag tagcggccgc tgcaggtcgt 2580

gactgggaaa accctggcga ctagtcttgg actcctgttg atagatccag taatgacctc 2640

agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga 2700

gaatccagac gttgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc 2760

atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca 2820

tattcaacgg gaaacgtctt gctcgaggcc gcgattaaat tccaacatgg atgctgattt 2880

atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt 2940

gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa 3000

tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac 3060

catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg 3120

gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc 3180

gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag 3240

cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc 3300

gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca 3360

taagcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa 3420

ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc 3480

agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt 3540

acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt 3600

tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca 3660

gagcattacg ctgacttgac gggacggcgg ctttgttgaa taaatcgaac ttttgctgag 3720

ttgaaggatc agatcacgca tcttcccgac aacgcagacc gttccgtggc aaagcaaaag 3780

ttcaaaatca ccaactggtc cacctacaac aaagctctca tcaaccgtgg ctccctcact 3840

ttctggctgg atgatggggc gattcaggcc tggtatgagt cagcaacacc ttcttcacga 3900

ggcagacctc agcgctattc tgaccttgcc atcacgactg tgctggtcat taaacgcgta 3960

ttcaggctga ccctgcgcgc tgcgcagggc tttattgatt ccatttttac actgatgaat 4020

gttccgttgc gctgcccgga ttacagccgg atcctctaga gtcgacctgc aggcatgctg 4080

atcggcacgt aagaggttcc aactttcacc ataatgaaat aagatcacta ccgggcgtat 4140

tttttgagtt atcgagattt tcaggagcta aggaagctaa aatgcgctca cgcaactggt 4200

ccagaacctt gaccgaacgc agcggtggta acggcgcagt ggcggttttc atggcttgtt 4260

atgactgtttttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg 4320

ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc 4380

ctaaaacaaa gttaaacatc atgagggaag cggtgatcgc cgaagtatcg actcaactat 4440

cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt 4500

acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg ctggttacgg 4560

tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt 4620

cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg 4680

acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc 4740

gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct 4800

tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct 4860

ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga 4920

actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt 4980

ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg 5040

agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac 5100

aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cactacgtga 5160

aaggcgagat caccaaggta gtcggcaaat aaactagtaa ataataaaaa agccggatta 5220

ataatctggc tttttatatt ctctgcataa ccctgcttcg gggtcattat agcgattttt 5280

tcggtatatc catccttttt cgcacgatat acaggatttt gccaaagggt tcgtgtagac 5340

tttccttggt gtatccaacg gcgtcagccg ggcaggatag gtgaagtagg cccacccgcg 5400

agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg cggtgctcaa cgggaatcct 5460

gctctgcgag gctggccgta ggccggccgc gatgcaggtg gctgctgaac ccccagccgg 5520

aactgacccc acaaggccct accggcgcgg cagcg 5739

<210> 42

<211> 1143

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene sequence of VCre recombinase

<400> 42

atgatcgaga accagctgag cctgctgggt gatttcagcg gcgtgcgtcc ggacgatgtt 60

aagaccgcga tccaggcggc gcaaaagaaa ggtattaacg ttgcggagaa cgaacaattc 120

aaagcggcgt ttgagcacct gctgaacgag ttcaagaaac gtgaggaacg ttacagcccg 180

aacaccctgc gtcgtctgga aagcgcgtgg acctgctttg tggattggtg cctggcgaac 240

catcgtcaca gcctgccggc gaccccggac accgttgagg cgttctttat cgaacgtgcg 300

gaggaactgc accgtaacac cctgagcgtg taccgttggg cgattagccg tgttcatcgt 360

gttgcgggtt gcccggaccc gtgcctggat atctatgtgg aggatcgtct gaaggcgatt 420

gcgcgtaaga aagtgcgtga gggcgaagcg gttaaacagg cgagcccgtt taacgaacaa 480

cacctgctga agctgaccag cctgtggtac cgtagcgaca aactgctgct gcgtcgtaac 540

ctggcgctgc tggcggtggc gtatgagagc atgctgcgtg cgagcgaact ggcgaacatc 600

cgtgttagcg acatggagct ggcgggtgat ggcaccgcga ttctgaccat cccgattacc 660

aagaccaacc acagcggcga gccggacacc tgcattctga gccaggatgt ggttagcctg 720

ctgatggact acaccgaagc gggcaagctg gacatgagca gcgatggttt cctgtttgtg 780

ggcgttagca aacacaacac ctgcatcaag ccgaagaaag ataaacagac cggtgaagtt 840

ctgcacaagc cgattaccac caaaaccgtg gagggcgttt tctatagcgc gtgggaaacc 900

ctggatctgg gtcgtcaagg cgtgaagccg tttaccgcgc acagcgcgcg tgttggtgcg 960

gcgcaggacc tgctgaagaa aggctacaac accctgcaaa tccagcaaag cggtcgttgg 1020

agcagcggcg cgatggttgc gcgttatggt cgtgcgatcc tggcgcgtga cggcgcgatg 1080

gcgcacagcc gtgtgaaaac ccgtagcgcg ccgatgcaat ggggcaagga cgagaaagat 1140

taa 1181

<210> 43

<211> 380

<212> PRT

<213> Artificial Sequence

<220>

<223> VCre recombinase

<400> 43

Met Ile Glu Asn Gln Leu Ser Leu Leu Gly Asp Phe Ser Gly Val Arg

1 5 10 15

Pro Asp Asp Val Lys Thr Ala Ile Gln Ala Ala Gln Lys Lys Gly Ile

20 25 30

Asn Val Ala Glu Asn Glu Gln Phe Lys Ala Ala Phe Glu His Leu Leu

35 40 45

Asn Glu Phe Lys Lys Arg Glu Glu Arg Tyr Ser Pro Asn Thr Leu Arg

50 55 60

Arg Leu Glu Ser Ala Trp Thr Cys Phe Val Asp Trp Cys Leu Ala Asn

65 70 75 80

His Arg His Ser Leu Pro Ala Thr Pro Asp Thr Val Glu Ala Phe Phe

85 90 95

Ile Glu Arg Ala Glu Glu Leu His Arg Asn Thr Leu Ser Val Tyr Arg

100 105 110

Trp Ala Ile Ser Arg Val His Arg Val Ala Gly Cys Pro Asp Pro Cys

115 120 125

Leu Asp Ile Tyr Val Glu Asp Arg Leu Lys Ala Ile Ala Arg Lys Lys

130 135 140

Val Arg Glu Gly Glu Ala Val Lys Gln Ala Ser Pro Phe Asn Glu Gln

145 150 155 160

His Leu Leu Lys Leu Thr Ser Leu Trp Tyr Arg Ser Asp Lys Leu Leu

165 170 175

Leu Arg Arg Asn Leu Ala Leu Leu Ala Val Ala Tyr Glu Ser Met Leu

180 185 190

Arg Ala Ser Glu Leu Ala Asn Ile Arg Val Ser Asp Met Glu Leu Ala

195 200 205

Gly Asp Gly Thr Ala Ile Leu Thr Ile Pro Ile Thr Lys Thr Asn His

210 215 220

Ser Gly Glu Pro Asp Thr Cys Ile Leu Ser Gln Asp Val Val Ser Leu

225 230 235 240

Leu Met Asp Tyr Thr Glu Ala Gly Lys Leu Asp Met Ser Ser Asp Gly

245 250 255

Phe Leu Phe Val Gly Val Ser Lys His Asn Thr Cys Ile Lys Pro Lys

260 265 270

Lys Asp Lys Gln Thr Gly Glu Val Leu His Lys Pro Ile Thr Thr Lys

275 280 285

Thr Val Glu Gly Val Phe Tyr Ser Ala Trp Glu Thr Leu Asp Leu Gly

290 295 300

Arg Gln Gly Val Lys Pro Phe Thr Ala His Ser Ala Arg Val Gly Ala

305 310 315 320

Ala Gln Asp Leu Leu Lys Lys Gly Tyr Asn Thr Leu Gln Ile Gln Gln

325 330 335

Ser Gly Arg Trp Ser Ser Gly Ala Met Val Ala Arg Tyr Gly Arg Ala

340 345 350

Ile Leu Ala Arg Asp Gly Ala Met Ala His Ser Arg Val Lys Thr Arg

355 360 365

Ser Ala Pro Met Gln Trp Gly Lys Asp Glu Lys Asp

370 375 380

<210> 44

<211> 2855

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic fragment 10

<400> 44

cacacaggaa acagctatga cctggattct caccaataaa aaacgcccgg cggcaaccga 60

gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc 120

caagctacga catcccggtg tgtagccgtt cgaccacgct gccgagcctg agatgctgct 180

cgtactcttg cagatccccg aagtcgatcg tgcgagtcag cccgccgcgg acgtcgaacg 240

tcagccgaac gttcatcgac cgaagccagg tgttctttgc cgcggtgtcc tgctcccgcc 300

accagtcccc gaaccgctgc ccggtctcgc gccactccca gccagacggg cgagcctcta 360

ggccctccag ctcctcttgc cgcgcggcca gcgccgcaat acgggcatcc agtgcttctc 420

gctgcggaga gccggcccgg taggccgggg agccgatcag cgacgtcagg tccaccagct 480

ccgcgttcac ctccgcgagt tcgaccgcgg agtccgagcc ggctacccag actttctcca 540

gacgctccgc gtccccgagc agatccagca cctgctcctc gcagaacgcg tcccactcgg 600

ccatcgccac cgtgccgttc ccgcagtgct tcgggaaccc catcgagcgg cagcggtagc 660

gcgggtgctt acgtcctccc ccggcgaact tgtacgcggg ctccccgcac accgcgcaga 720

acaacacccg cagcagcagc gacggggtag acaccgcggg cttcgcccgg gaggtcttca 780

cgagctcggc gcgcagcgcc tccagctgct cacgggtcag gatcggctca gcccgcacca 840

gcggggctcc gtcgtcgtct cggacggtct taccgttcag agtcgcgtac ccgagcatcg 900

cctcggagat catcgatcgc ttcagcgcgg tagccgacca ctcccggccc tgcggctcgc 960

ggccttgcag ctgcgcgaag tagtccttcg gcgacaggac accacgccgg ttcaggtcgt 1020

gggccaccag gtgcagcggc tcgtggttgt cgacgacgcg gtgatacacc tcgaggatgc 1080

gctctcgctg cacagggtcc ggcaccagcc gccactcccc gtccacgcgc gtaggcaggt 1140

atccccacgg cggcagggat cctcggtatt tcccggcgcg gatattgaaa tgcgcagccg 1200

aacggttccg ctctttgatc gcttctaatt ccatctgcgc caccgttccc ataagcgcga 1260

tgacgaccgc cgcaaacggc gtcgtcgtat cgaagtgcgc ttcggtcgcg gagacgacca 1320

gcttcttgtg gtcctcggcc cagtggacca gctgttgcag atgccggatc gatcgggtca 1380

accggtctac ccggtacgcc acgatcacgt cgaacggttg ctcctcgaac gctagccacc 1440

gggccaggtt cggtctgcgc ttccggtcga acggatcgac cgccccggag acgtccagat 1500

cctccgctac cccgacgacg tcccagccgc gctgggcgca gagctgctgg caagactcca 1560

gctgacgctc cggtgaagtc gtagcatcgg tgacgcggga caggcggatg actaccaggg 1620

ctctcatcta gtatttctcc tctttctcta gtattaaaca aaattatttg tagaggctgt 1680

ttcgtcctca cggactcatc agaccggaaa gcacatccgg tgacagcttg ctcgcaggtc 1740

aaagggtata ctgggattcc agtgaacgca atcaatttct gagaactgtc attctcggaa 1800

attgagggtt tgtaccgtac accactgaga ccgcggtggt tgaccagaca aaccacgagg 1860

gagaccagaa acaaaaaaag gccccccgtt agggaggcct tcaataattg gttatcattt 1920

gtacagttca tccataccat gcgtgatgcc cgctgcggtt acgaactcca gcagaaccat 1980

atgatcgcgt ttctcgttcg gatctttaga cagaacgctt tgcgtgctca gatagtgatt 2040

gtctggcagc agaacaggac catcaccgat tggagtgttt tgctggtagt gatcagccag 2100

ctgcacgctg ccatcctcca cgttgtggcg aattttaaaa ttcgctttaa tgccattttt 2160

ttgtttatcg gcggtgatgt aaacattgtg gctgttaaaa ttgtattcca gcttatggcc 2220

caggatattg ccgtcttctt taaagtcaat gcctttcagc tcaatgcggt ttaccagggt 2280

atcgccttca aatttcactt ccgcacgcgt tttgtacgtg ccgtcatcct taaaggaaat 2340

cgtgcgttcc tgcacatagc cttccggcat ggcggacttg aagaagtcat gctgcttcat 2400

atggtccgga taacgagcaa agcactgaac accataagtc agcgtcgtta ccagagtcgg 2460

ccaaggtacc ggcagtttac cagtagtaca gatgaacttc agcgtcagtt taccattagt 2520

tgcgtcacct tcaccctcgc cacgcacgga aaacttatga ccgttgacat caccatccag 2580

ttccaccaga atagggacga caccagtgaa cagctcttcg cctttacgca tctagtattt 2640

ctcctctttc tctagtaact cttaaacaaa attatttgta gaggctgttt cgtcctcacg 2700

gactcatcag accggaaagc acatccggtg acagcttgct cgcaggtcaa aatatatact 2760

gggattccag tgaacgcaac aggatgtgac gagcggtgtg gtcaatttct gagaactgtc 2820

attctcggaa attgaactgg ccgtcgtttt acaac 2949

<210> 45

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Sequence of VloxP

<400> 45

tcaatttccg agaatgacag ttctcagaaa ttga 34

<210> 46

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 46

tcggcggcgg ccgggcgtg 19

<210> 47

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 47

caccgattgg agtgttttgc 20

Claims (8)

1. A gene insertion method based on a site-specific recombinase suitable for Euaerobes rosenbergii, comprising the following steps: 1) Inserting the attB site corresponding to the site-specific recombinase Bxb1 into the genome of Eutropha rosenbergii to construct a recombinant Eutropha rosenbergii with the attB sequence integrated in the genome, wherein the Eutropha rosenbergii is Ralstonia eutrophaH16; 2) Constructing a recombinant vector containing an exogenous sequence, a site-specific recombinase Bxb1 gene, and an attP sequence corresponding to the site-specific recombinase Bxb1, the recombinant vector is a plasmid vector that cannot replicate in Euaeobes rosenbergii, and The backbone part of the recombinant vector contains the replicon pMB1 that can replicate in Escherichia coli S17-1 but cannot replicate in E. Because of antibiotic resistance genes; 3) Transform the recombinant vector constructed in step 2) into Escherichia coli S17-1, and then transfer it into the recombinant Euaerobes rosenbergii constructed in step 1) by conjugative transformation method, using the site-specific Sexual recombinase Bxb1 mediates the recombination between the attB and attP sequences, thereby integrating the recombinant vector into the genome of the recombinant Euaeobesia rosenbergii, Wherein, in step 1), the sequence of the attB site corresponding to the site-specific recombinase Bxb1 is shown in SEQ ID NO: 10; in step 2), the amino acid sequence of the site-specific recombinase Bxb1 is shown in As shown in SEQ ID NO: 21, the attP sequence corresponding to the site-specific recombinase Bxb1 is shown in SEQ ID NO: 22. 2. The method according to claim 1, wherein, in step 2), the antibiotic resistance gene is selected from kanamycin resistance gene, tetracycline resistance gene, streptomycin resistance gene and spectinomycin resistance gene One or more of the sex genes. 3. The method according to claim 1, wherein, in step 2), the nucleotide sequence of the site-specific recombinase Bxb1 gene is shown in SEQ ID NO: 20. 4. A method for gene insertion based on a site-specific recombinase suitable for Euaerobes rosenbergii, comprising the following steps: a) Inserting the attB site corresponding to the site-specific recombinase Bxb1 into the genome of Eutropha rosenbergii to construct a recombinant Eutropha rosenbergii with the attB sequence integrated in the genome, wherein the Eutropha rosenbergii is Ralstonia eutrophaH16; b) Constructing a recombinant vector containing the VCre recombinase gene whose amino acid sequence is shown in SEQ ID NO: 43, the recombinant vector in this step is a plasmid vector capable of replicating in Euaerobes rosenbergii, and the backbone part of the recombinant vector Contains the replicon pBBR1 capable of replicating both in Escherichia coli S17-1 and in E. rosenbergii, and the backbone part of the recombinant vector in this step also contains a selectable marker gene, which is antibiotic resistance Gene; c) Construct a recombinant vector containing foreign sequence, site-specific recombinase Bxb1 gene, attP sequence corresponding to site-specific recombinase Bxb1, and two VloxP sequences that can be specifically recognized by VCre recombinase in step b). , wherein, the foreign sequence and the attP sequence are between the two VloxP sequences, the site-specific recombinase Bxb1 gene is not between the two VloxP sequences, and the recombinant vector in this step is A plasmid vector that cannot be replicated in Euaeobes rosenbergii, and the backbone part of the recombinant vector in this step contains the replicon pMB1 that can replicate in E. The backbone part of the recombinant vector also includes a selection marker gene, and the selection marker gene is an antibiotic resistance gene; d) Transform the recombinant vector constructed in step c) into Escherichia coli S17-1, and then transfer it into the recombinant Euaeobesia rosenbergii constructed in step a) by conjugative transformation method, using the site-specific The recombinase Bxb1 mediates the recombination between the attB and attP sequences, thereby integrating the recombinant vector in step c) into the genome of the recombinant Euaerobes rosenbergii; e) Transform the recombinant vector constructed in step b) into Escherichia coli S17-1, and then transfer to step d) by conjugative transformation method. The recombinant Roche True Oxygen that integrates the recombinant vector in step c) into the genome obtained bacteria, thereby deleting the backbone part of the recombinant vector described in step c) from the genome of the recombinant Euaeobes rosenbergii, Wherein, in step a), the sequence of the attB site corresponding to the site-specific recombinase Bxb1 is shown in SEQ ID NO: 10; in step c), the amino acid sequence of the site-specific recombinase Bxb1 gene As shown in SEQ ID NO: 21, the attP sequence corresponding to the site-specific recombinase Bxb1 is shown in SEQ ID NO: 22, and the VloxP sequence is shown in SEQ ID NO: 45. 5. The method according to claim 4, wherein, in step b), the antibiotic resistance gene is selected from kanamycin resistance gene, tetracycline resistance gene, streptomycin resistance gene and spectinomycin resistance gene One or more of the sex genes. 6. The method according to claim 4, wherein, in step b), the nucleotide sequence of the VCre recombinase is shown in SEQ ID NO: 42. 7. The method according to claim 4, wherein, in step c), the antibiotic resistance gene is selected from kanamycin resistance gene, tetracycline resistance gene, streptomycin resistance gene and spectinomycin resistance gene One or more of the sex genes. 8. The method according to claim 4, wherein, in step c), the nucleotide sequence of the site-specific recombinase Bxb1 gene is shown in SEQ ID NO: 20.

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