CN113355345A - Method for integrating exogenous sequence into genome - Google Patents

Abstract

The invention provides a method for integrating exogenous sequences into a genome. The method comprises the following steps: 1) inserting an attB site corresponding to a site-specific recombinase Bxb1 into the genome of the Eubacterium rolfsii to construct a recombinant Eubacterium rolfsii with an attB sequence integrated on 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 Bxb 1; 3) transferring the recombinant vector constructed in the step 2) into the recombinant Rogowski eubacterium constructed in the step 1), and mediating recombination between attB and attP sequences by using the site-specific recombinase Bxb1, thereby integrating the recombinant vector into the genome of the recombinant Rogowski eubacterium.

Description

Method for integrating exogenous sequence into genome

Technical Field

The invention belongs to the technical field of biology, and relates to a method for integrating a foreign sequence into a genome, in particular to a gene insertion method based on a site-specific recombinase, which is suitable for an oxygen bacterium rolfsii.

Background

The eubacterium rolfsii (Ralstonia eutropha, also called cupriavidius dicator) is an important model bacterium for researching the synthesis of PHA (polyhydroxyalkanoate), which is a completely degradable bio-based material, and has the potential to be used as a strain for PHA industrial production.

The existing gene editing technical means of the eubacterium rolfsii is based on homologous recombination taking suicide plasmid as a vector. The suicide plasmid contains R6K gamma replicon which can not replicate in the eubacterium rolfsii, resistance gene and DNA sequence (called homology arm) for homologous recombination, and the sequence of the homology arm can be freely designed according to the gene site needing editing. After the suicide plasmid is transferred into the oxygen bacterium rochei, homologous recombination occurs between a homologous arm sequence on the suicide plasmid vector and the same sequence on a genome under the selective pressure of antibiotics, so that site-directed gene editing is realized. In addition, a gene editing method based on CRISPR/Cas9 has been developed in Eubacterium rolfsii, the principle is also homologous recombination, and the selection pressure is changed to that the Cas9 protein is used for site-specific Genome cutting (Xiong, B., Li, Z., Liu, L., ZHao, D., Zhang, X., Bi, C.,2018.Genome editing of Ralstonia eutropha usage an electrophoresis-based CRISPR-Cas9technique. Biotechnol. Biofuels.11: 172).

Homologous recombination is the recombination that occurs between non-sister chromatids or between or within DNA molecules containing homologous sequences on the same chromosome. Homologous recombination relies on the presence of homologous regions or sequences, where DNA strand breaks are random, exposing sequences that bind to a range of proteins (e.g., RecA in prokaryotic cells, and Rad51 in eukaryotic cells, etc.), ultimately allowing cross-recombination between or within DNA molecules. However, the length of the integrated exogenous sequence is limited by the random sequence fragmentation and the recombination system of the underpinning microorganism, and in addition, homologous regions (such as the length and the position of the homologous sequence) need to be optimized to achieve better gene editing effect.

Although homologous recombination can theoretically realize gene knockout, insertion and replacement, in practical application, the larger the inserted fragment is, the smaller the gene insertion efficiency is, and the application in scenes such as large fragment gene insertion of multiple gene lines, complex metabolic pathways and the like is limited.

In contrast, site-specific recombination, during recombination, relies not on the homology of the DNA sequences (although partly on the short homologous sequences) but on the presence of short DNA sequences capable of binding to certain enzymes, and the cleavage and religation of the DNA strands occurs under the action of these recombinases with high efficiency and specificity. Therefore, the site specificity is realized, the high specificity and the high conservation of the recombination are ensured, and the efficiency of the site-specific recombination is superior to that of a homologous recombination method depending on proteins such as RecA and the like.

The site-specific recombination system has the advantages of high efficiency, controllability, accuracy, rapidness, strong specificity, good element orthogonality and the like, has wide application prospect, and can be applied to gene editing and genetic engineering operation. The system is derived from a bacteriophage which inserts its DNA into the chromosome of a host bacterium when infecting the bacterium and replicates and divides in synchrony with the host, and this process requires site-specific recombinases within the phage to recognize specific "recognition sites" - "attP" and "attB" (attP and attB are present in the phage and host chromosome respectively). The recombinase mediates recombination between the two sequences attP and attB, which inserts the phage DNA into the host bacterium chromosome, and the original attP and attB sequences form two new sequences "attL" and "attR" on the chromosome. As a tool, the site-specific recombination system consists of a recognition site/sequence of integrase and integrase that recognizes DNA sequences and mediates DNA recombination. By changing the position and sequence orientation of the recognition sites of the recombinase, the site-specific recombinase can serve as a tool enzyme for inserting a specific sequence into a DNA strand or deleting or inverting a specific sequence from a DNA strand, and thus is widely used for any incision-making cutting and suturing operation (recombination of DNA fragments) in various underplate microorganisms.

Site-specific recombination-based systems have been applied to various microorganisms, even eukaryotic cells, including E.coli, Bacillus, Saccharomyces cerevisiae, T cells, and even embryonic stem cells, and are widely used for integration of foreign genes, production of natural products, gene therapy, and the like. For example, integration of a 34kb sized metabolic pathway in the E.coli genome for alginate degradation and ethanol production can be achieved using integrase as a gene editing tool (Christine Nicole S. Santos., draw D. Regitsky., and Yasuo Yoshikuni.,2013. augmentation of stable and complex biological systems through restriction enzyme engineering. Nature communications.4: 2503).

In conclusion, the existing gene editing technical means of the eubacterium rolfsii is based on homologous recombination taking suicide plasmids as vectors and a gene editing method based on CRISPR/Cas 9. Homologous recombination and gene editing methods of CRISPR/Cas9 become less efficient at inserting genes as the insert becomes longer. The site-specific recombinase can realize efficient long fragment insertion, and the site-specific recombinase and related tools thereof available in the eubacterium rolfsii are not available at present.

Disclosure of Invention

Aiming at the defects of the prior art, the inventor develops a gene insertion method based on site-specific recombinase suitable for the Roche bacteria, and perfects the Roche bacteria gene editing technology by adding the existing homologous recombination technology.

Accordingly, in one aspect, the present invention provides a method for inserting a gene based on a site-specific recombinase into eubacterium rolfsii, comprising the steps of:

1) inserting an attB site corresponding to a site-specific recombinase Bxb1 into the genome of the Eubacterium rolfsii to construct a recombinant Eubacterium rolfsii with an attB sequence integrated on 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 Bxb 1;

3) transferring the recombinant vector constructed in the step 2) into the recombinant Rogowski eubacterium constructed in the step 1), and mediating recombination between attB and attP sequences by using the site-specific recombinase Bxb1, thereby integrating the recombinant vector into the genome of the recombinant Rogowski eubacterium.

By the method, the exogenous sequence (DNA fragment) and the vector can be integrated on the genome of the recombinant bacterium. In practice, it is preferred that the vector portion other than the DNA fragment to be integrated be deleted as necessary.

Therefore, the method for inserting a gene based on a site-specific recombinase suitable for Eubacterium rolfsii of the present invention preferably comprises the steps of:

a) inserting an attB site corresponding to a site-specific recombinase Bxb1 into the genome of the Eubacterium rolfsii to construct a recombinant Eubacterium rolfsii with an attB sequence integrated on the genome;

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

c) constructing a recombinant vector containing an exogenous sequence, a site-specific recombinase Bxb1 gene, attP sequence corresponding to the site-specific recombinase Bxb1, and 2 VloxP sequences capable of being specifically recognized by VCre recombinase in step b), wherein the exogenous sequence and the attP sequence are between the 2 VloxP sequences, and the site-specific recombinase Bxb1 gene is not between the 2 VloxP sequences;

d) transferring the recombinant vector constructed in the step c) into the recombinant Rogowski bacterium constructed in the step a), and mediating recombination between attB and attP sequences by using the site-specific recombinase Bxb1, thereby integrating the recombinant vector into the genome of the recombinant Rogowski bacterium;

e) transferring the recombinant vector constructed in the step b) into the recombinant bacterium integrated with the recombinant vector on the genome obtained in the step d), so as to delete the skeleton part of the recombinant vector from the genome.

In step 1) or a) of the process according to the invention, the said Eubacterium rolfsii may also be another bacterium of the genus Ralstonia. Preferably, the eubacterium rolfsii 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 incapable of replication in Eubacterium rolfsii, such as a suicide plasmid. Preferably, the backbone portion of the recombinant vector comprises a replicon capable of replicating in E.coli (e.g., S17-1) but incapable of replicating in Eubacterium rolfsii, preferably the replicon is one or more selected from the group consisting of a pMB1 replicon, a pUC replicon, a p15a replicon and a R6K γ replicon, more preferably the replicon is a pMB1 replicon. Preferably, the backbone portion of the recombinant vector further comprises a selectable marker gene, such as an antibiotic resistance gene; more preferably, the backbone portion of the recombinant vector further comprises an antibiotic resistance gene, particularly, the antibiotic resistance gene is one or more selected from the group consisting of a kanamycin resistance gene, a tetracycline resistance gene, a streptomycin resistance gene, and a spectinomycin resistance gene, and more particularly, the antibiotic resistance gene is a kanamycin resistance gene. More preferably, the plasmid vector may be derived from the plasmid pK18 mobsacB.

In step b) of the method of the present invention, preferably, the recombinant vector may be a plasmid vector capable of replicating in Eubacterium rolfsii. Preferably, the backbone portion of the recombinant vector comprises a replicon capable of replicating both in E.coli (e.g., S17-1) and in Eubacterium rolfsii, preferably the replicon is one or more selected from the group consisting of a pBBR1 replicon, an SC101 replicon, and a RK2 replicon, more preferably the replicon is a pBBR1 replicon. Preferably, the backbone portion of the recombinant vector further comprises a selectable marker gene, such as an antibiotic resistance gene; more preferably, the backbone portion of the recombinant vector further comprises an antibiotic resistance gene, preferably, the antibiotic resistance gene is one or more selected from the group consisting of kanamycin resistance gene, tetracycline resistance gene, streptomycin resistance gene, and spectinomycin resistance gene, and more preferably, the antibiotic resistance gene is kanamycin and spectinomycin resistance gene. More preferably, the plasmid vector may be derived from plasmid pBBR1MCS 2.

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

In step 2) or c) of the method of the present invention, there are no particular requirements or restrictions on the exogenous sequence. For example, the length of the foreign sequence may be 1,000,000bp or less, preferably 100,000bp or less.

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 shown as 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 shown as 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 shown as SEQ ID NO. 22.

In step b) of the method of the present invention, preferably, the amino acid sequence of the VCre recombinase is 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 represented by SEQ ID NO 41.

In step c) of the method of the present invention, preferably, said VloxP sequence is represented by 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 rhodobacter rolfsii by a conjugative transformation method, and the recombinant rhodobacter rolfsii having the recombinant vector integrated on its genome is selected by using a selection marker, using the property that the suicide plasmid cannot replicate in the host bacterium.

Further preferably, step 1) or a) of the method of the invention can be carried out using methods known in the art (Xiong, B., Li, Z., Liu, L., ZHao, D., Zhang, X., Bi, C.,2018.Genome editing of Ralstonia eutropha usage an electrophoresis-based CRISPR-case 9technique. Biotechnol. Biofuels.11: 172). For example, a suicide plasmid in which attB site is located between two homologous fragments and cannot be replicated in a Roche mycorrhizal fungi is constructed, the suicide plasmid is transferred into the Roche mycorrhizal fungi, and a recombinant Roche mycorrhizal fungi integrated with attB sequence is obtained by screening by utilizing the characteristic that the suicide plasmid cannot be replicated in a host bacterium. In particular, step 1 or a) of the process of the invention comprises the following steps: carrying out PCR amplification by using SEQ ID NO 1 and SEQ ID NO 2 as primers and a Ralstonia eutropha H16 genome as a template to obtain homologous fragments H1 and H2, carrying out PCR amplification by using SEQ ID NO 3 and SEQ ID NO 4 as primers and a plasmid pK18mobsacB as a template to obtain a carrier fragment, carrying out amplification by using SEQ ID NO 7 and SEQ ID NO 8 as primers and a SEQ ID NO 9 as a template to obtain a fragment containing an attB sequence corresponding to Bxb1, and connecting the H1, H2 and attB sequence with the carrier fragment by a Gibson Assemby method, wherein the attB site is positioned between the two homologous fragments H1 and H2 to obtain a recombinant plasmid pK18mobsacB-Bxb 1; transferring the recombinant plasmid pK18mobsacB-Bxb1 into escherichia coli S17-1, transferring the recombinant plasmid into Ralstonia eutropha H16 by a joint transformation method, screening a positive clone by using an LB (Langmuir-Blodgett) plate simultaneously containing kanamycin and apramycin by utilizing the characteristic that a suicide plasmid cannot be copied in host bacteria, and integrating the recombinant plasmid with a homologous fragment in the positive clone into specific positions of H1 and H2 on a genome to obtain a first homologous recombinant bacterium; the first homologous recombinant strain is subjected to single clone culture on an LB plate containing cane sugar, clones without kanamycin resistance are screened from the single clones, and a recombinant strain with an attB sequence of Bxb1 integrated into a genome is identified, wherein the obtained recombinant strain is Ralstonia eutropha Bxb 1-attB.

Further preferably, step 2) of the process of the invention comprises the following steps: PCR amplification is carried out by taking SEQ ID NO 5 and SEQ ID NO 6 as primers and plasmid pK18mobsacB as a template to obtain a vector fragment, amplification is carried out by taking SEQ ID NO 9and SEQ ID NO 10 as primers and SEQ ID NO 19 as a template to obtain a fragment containing Bxb1 recombinase gene and corresponding attP sequence, and the fragment is connected with the vector fragment by a Gibson Assembly method to obtain recombinant plasmid pBxb 1-attP.

Further preferably, step 3) of the method of the invention comprises the following steps: transferring the recombinant plasmid pBxb1-attP into escherichia coli (such as S17-1), transferring the recombinant plasmid pBxb1-attP into Ralstonia eutropha Bxb1-attB by a conjugative transformation method, and screening by using a screening marker to obtain the recombinant eubacterium rolfsii with the recombinant vector integrated on a genome by utilizing the characteristic that a suicide plasmid cannot be replicated in host bacteria.

Further preferably, step b) of the process of the invention comprises the following steps: PCR amplification is carried out by using SEQ ID NO. 36 and SEQ ID NO. 37 as primers and plasmid pBBR1MCS2 as a template to obtain a replicon fragment; amplifying by using SEQ ID NO 38 and SEQ ID NO 39 as primers and SEQ ID NO 40 as a template to obtain a DNA fragment, wherein the DNA fragment contains a VCre recombinase gene, a kanamycin resistance gene and a spectinomycin resistance gene; the fragment was ligated to the replicon fragment by the Gibson Assembly method to obtain a recombinant plasmid pVCre.

Further preferably, step c) of the process of the invention comprises the following steps: PCR amplification is carried out by using SEQ ID NO. 5 and SEQ ID NO. 6 as primers and plasmid pK18mobsacB as a template to obtain a vector fragment; obtaining a fragment containing Bxb1 recombinase gene and corresponding attP sequence, a foreign gene to be integrated and 2 VloxP sequences which can be specifically recognized by VCre recombinase, and connecting the fragment with a vector fragment by a Gibson Assembly method to obtain a recombinant plasmid. Specifically, for example, the exogenous gene is GFP, the step c) of the method of the present invention comprises the following steps: PCR amplification is carried out by using SEQ ID NO. 5 and SEQ ID NO. 6 as primers and plasmid pK18mobsacB as a template to obtain a vector fragment; amplifying by using SEQ ID NO. 17 and SEQ ID NO. 18 as primers and SEQ ID NO. 43 as a template to obtain a DNA fragment containing Bxb1 recombinase gene and corresponding attP sequence, a foreign Gene (GFP) to be integrated and 2 VloxP sequences capable of being specifically recognized by VCre recombinase; the fragments were ligated to the vector fragment by the Gibson Assembly method to give the recombinant plasmid pBxb 1-attP-VCre.

Further preferably, step d) of the process according to the invention comprises the following steps: transferring the recombinant plasmid in the step c), such as pBxb1-attP-VCre, into Escherichia coli S17-1, transferring the recombinant plasmid into Ralstonia eutropha Bxb1-attB by a conjugative transformation method, and screening by a screening marker to obtain the recombinant eubacterium rolfsii with the recombinant vector integrated on the genome.

Further preferably, step e) of the process of the invention comprises the steps of: transferring the recombinant plasmid pVCre obtained in the step b) into Escherichia coli S17-1, transferring the recombinant strain obtained in the step d) by a joint transformation method, and screening by a screening marker to obtain a recombinant strain (such as Ralstonia eutropha Bxb1-GFP) with an exogenous gene integrated into a genome.

Preferably, step e) of the process of the invention further comprises: and (3) culturing the recombinant bacteria with the exogenous genes integrated into the genome on a non-resistant plate so that the recombinant bacteria lose the recombinant vector containing the VCre recombinase gene.

In the gene insertion method based on the site-specific recombinase applicable to the eubacterium rolfsii, as shown in figure 1: firstly, inserting an attB site corresponding to a site-specific recombinase Bxb1 into the genome of the Eubacterium rolfsii to construct a recombinant Eubacterium rolfsii with an attB sequence integrated on the genome; constructing a recombinant vector containing a VCre recombinase gene; constructing a recombinant vector containing a foreign sequence, a site-specific recombinase Bxb1 gene, an attP sequence corresponding to the site-specific recombinase Bxb1, and 2 VloxP sequences specifically recognized by the VCre recombinase, wherein said exogenous sequence and said attP sequence are between said 2 VloxP sequences, the gene of the site specific recombinase Bxb1 is not between the 2 VloxP sequences (as shown in FIG. 2), transferring the recombinant vector into the recombinant Roche bacterium integrated with attB sequence on the constructed genome, mediating recombination between attB and attP sequences using the site-specific recombinase Bxb1, thereby integrating the recombinant vector onto the genome of the recombinant Rogowski Eubacterium, wherein, the recombinase mediates recombination between two sequences attP and attB, the process inserts phage DNA into a host bacterium chromosome, and the original attP and attB sequences form two new sequences 'attL' and 'attR' on the chromosome; subsequently, the recombinant vector containing the VCre recombinase gene constructed as described above is transferred into a recombinant bacterium having the recombinant vector integrated into its genome, and the backbone portion of the recombinant vector is deleted from the genome.

In another aspect, the present invention provides a recombinant vector (suicide plasmid) comprising an exogenous sequence, a gene of site-specific recombinase Bxb1, and an attP sequence corresponding to site-specific recombinase Bxb 1. Preferably, said recombinant vector further comprises 2 VloxP sequences capable of being specifically recognized by VCre recombinase, wherein said exogenous sequence and said attP sequence are between said 2 VloxP sequences, and said site-specific recombinase Bxb1 gene is not between said 2 VloxP sequences. Preferably, the backbone portion of the recombinant vector comprises a replicon capable of replicating in E.coli (e.g., S17-1) but incapable of replicating in Eubacterium rolfsii, preferably the replicon is one or more selected from the group consisting of a pMB1 replicon, a pUC replicon, a p15a replicon and a R6K γ replicon, more preferably the replicon is a pMB1 replicon. The backbone portion 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, and more preferably, the antibiotic resistance gene is a kanamycin resistance gene. Preferably, the amino acid sequence of the site-specific recombinase Bxb1 gene is shown in SEQ ID NO: 20. More preferably, the nucleotide sequence of the site-specific recombinase Bxb1 gene is shown in SEQ ID NO: 21. Preferably, the attP sequence corresponding to the site-specific recombinase Bxb1 is shown as SEQ ID NO 22. Preferably, the VloxP sequence is shown in SEQ ID NO: 44.

In yet 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 capable of replicating both in E.coli (e.g., S17-1) and in Eubacterium rolfsii, preferably the replicon is one or more selected from the group consisting of a pBBR1 replicon, an SC101 replicon and a RK2 replicon, more preferably the replicon is a pBBR1 replicon. The backbone portion 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, and more preferably, the antibiotic resistance gene is a kanamycin and spectinomycin resistance gene. Preferably, the amino acid sequence of the VCre recombinase is shown as 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, and can realize the high-efficiency integration of an exogenous sequence into a genome.

Drawings

FIG. 1 is a schematic diagram of the process of the present invention for integrating a gene mediated by a site-specific recombinase.

FIG. 2 is a schematic diagram showing the structure of a vector containing a foreign sequence, a recombinase and an attP site according to the present invention.

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

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

Detailed Description

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

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The enzymatic reagents used were purchased from ThermoFisher and New England Biolabs (NEB), the kit for plasmid extraction was purchased from Tiangen Biotechnology technology (Beijing) Ltd, the kit for DNA fragment recovery was purchased from omega USA, the corresponding procedures were performed strictly according to the product instructions, and all media were prepared with deionized water if no special instructions were given.

The formula of the culture medium is as follows:

LB culture medium: 5g/L yeast extract (from OXID, U.K., catalog No. LP0021), 10g/L peptone (from OXID, U.K., catalog No. LP0042),10g/L NaCl, and the balance water. Adjusting pH to 7.0-7.2, and sterilizing with high pressure steam.

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

Example 1: construction of recombinant Eubacterium rolfstromia bacterium Ralstonia eutropha Bxb1-attB incorporating attB sequence

PCR amplification was performed using Ralstonia eutropha H16 (purchased from China general microbiological culture Collection center, CGMCC 1.7092) genome as template to obtain homologous fragments H1 and H2, and plasmid pK18mobsacB (Orita, I., Iwazawa, R., Nakamura, S., Fukui, T.2012)An Identification of mutation sites in the replication of nucleic acid and amplification of cleavage activity in the wild strain H16 for polyhydroxyalkanoate production.J.biosci.Bioeng.113,63-69) as a template to obtain a vector fragment, and a fragment containing the attB sequence corresponding to Bxb1 was obtained by amplification using the primer as a template, according to the commercial kit (Gibson @) (Gibson @ cleavage-cleavage

Master Mix, purchased from New England Biolabs (NEB) Inc.) ligated H1, H2 and attB sequences to the vector fragment by the Gibson Assembly method to yield recombinant plasmid pK18mobsacB-Bxb 1. The primers used are as follows:

the sequence of synthetic fragment 01 was:

GGCAGAGAGACAATCAAATCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCCAGGAAACAGCTATGACGGTTCGGCCGGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCATCCGGGCACTGGCCGTCGTTTTACAACCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGCCTGCCGGCCTGGTTCAAC(SEQ ID NO:9)

wherein the corresponding attB sequence of Bxb1 is as follows:

TCGGCCGGCTTGTCGACGACGGCGGTCTCCGTCGTCAGGATCATCCGGGC(SEQ ID NO:10)

the recombinant plasmid pK18mobsacB-Bxb1 was transformed into E.coli S17-1(ATCC No. 47055, available from American Type Culture Collection) and then into Ralstonia eutropha H16 by the conjugative transformation method, and positive clones were selected using LB plates containing both 200. mu.g/ml kanamycin and 100. mu.g/ml apramycin, taking advantage of the inability of suicide plasmids to replicate in host bacteria. The recombinant plasmid with homologous fragment in the positive clone is integrated into the genome at the specific positions of H1 and H2, and is the first homologous recombinant bacterium.

The first homologous recombinant strain is subjected to single colony culture on an LB plate containing 100mg/ml of sucrose, clones without kanamycin resistance are screened out from the single colonies, and a recombinant strain with an attB sequence of Bxb1 integrated into a genome is identified by PCR (polymerase chain reaction) by using primers primer 7 and primer8, and the obtained recombinant strain is Ralstonia eutropha Bxb 1-attB.

Comparative example 1: construction of recombinant Eubacterium rolfsii Ralstonia eutropha PhiC31-attB incorporating attB sequence

Carrying out PCR amplification by taking Ralstonia eutropha H16 genome as a template to obtain homologous fragments H1 and H2, carrying out PCR amplification by taking plasmid pK18mobsacB as a template to obtain a vector fragment, carrying out PCR amplification by taking a synthetic fragment 02 as a template by using primers to obtain a fragment containing an attB sequence corresponding to PhiC31, and carrying out PCR amplification according to a commercial kit (Gibson)

Master Mix, purchased from New England Biolabs (NEB) Inc.) ligated H1, H2 and attB sequences to the vector fragment by the Gibson Assembly method to give recombinant plasmid pK18mobsacB-PhiC 31. The primers used are as follows:

the sequence of synthetic fragment 02 was:

GGCAGAGAGACAATCAAATCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCCAGGAAACAGCTATGACGGTCGCGCCCGGGGAGCCCAAGGGCACGCCCTGGCACACTGGCCGTCGTTTTACAACCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGCCTGCCGGCCTGGTTCAAC(SEQ ID NO:11)

wherein the corresponding attB sequence of PhiC31 is as follows:

CGCGCCCGGGGAGCCCAAGGGCACGCCCTGGCAC(SEQ ID NO:12)

the recombinant plasmid pK18mobsacB-PhiC31 is transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha H16 by a conjugation transformation method, and positive clones are screened by using an LB plate simultaneously containing 200 mug/ml kanamycin and 100 mug/ml apramycin by utilizing the characteristic that the suicide plasmid cannot replicate in host bacteria. The recombinant plasmid with homologous fragment in the positive clone is integrated into the genome at the specific positions of H1 and H2, and is the first homologous recombinant bacterium.

The first homologous recombinant strain is subjected to single colony culture on an LB plate containing 100mg/ml of sucrose, clones without kanamycin resistance are screened out from the single colonies, and a recombinant strain with the integrated genome of the attB sequence of PhiC31 is identified by PCR through primers primer 7 and primer8, and the obtained recombinant strain is Ralstonia eutropha PhiC 31-attB.

Comparative example 2: construction of recombinant Ralstonia eutropha TP901-attB integrating attB sequence

Carrying out PCR amplification by taking a Ralstonia eutropha H16 genome as a template to obtain homologous fragments H1 and H2, carrying out PCR amplification by taking a plasmid pK18mobsacB as a template to obtain a vector fragment, carrying out PCR amplification by taking a synthetic fragment 03 as a template to obtain a fragment containing an attB sequence corresponding to TP901 by using a primer, and carrying out PCR amplification according to a commercial kit (Gibson)

Master Mix, purchased from New England Biolabs (NEB) Inc.) ligated H1, H2 and attB sequences to the vector fragment by the Gibson Assembly method to give recombinant plasmid pK18mobsacB-TP 901. The primers used are as follows:

the sequence of synthetic fragment 03 was:

GGCAGAGAGACAATCAAATCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCCAGGAAACAGCTATGACGGTTATGCCAACACAATTAACATCTCAATCAAGGTAAATGCTTTTTGCTTTTTTTGACTGGCCGTCGTTTTACAACCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGCCTGCCGGCCTGGTTCAAC(SEQ ID NO:13)

wherein, the attB sequence corresponding to TP901 is:

TATGCCAACACAATTAACATCTCAATCAAGGTAAATGCTTTTTGCTTTTTTTG(SEQ ID NO:14)

the recombinant plasmid pK18mobsacB-TP901 is transferred into Escherichia coli S17-1, then transferred into Ralstonia eutropha H16 by a conjugative transformation method, and positive clones are screened by using an LB plate simultaneously containing 200 mug/ml kanamycin and 100 mug/ml apramycin by utilizing the characteristic that the suicide plasmid cannot be replicated in host bacteria. The recombinant plasmid with homologous fragment in the positive clone is integrated into the genome at the specific positions of H1 and H2, and is the first homologous recombinant bacterium.

The first homologous recombinant strain is subjected to single colony culture on an LB plate containing 100mg/ml of sucrose, clones without kanamycin resistance are screened out from the single colonies, primers 7 and 8 are used for carrying out PCR (polymerase chain reaction) to identify a recombinant strain with an attB sequence of TP901 integrated into a genome, and the obtained recombinant strain is Ralstonia eutropha TP 901-attB.

Comparative example 3: construction of recombinant Eubacterium rolfstonia eutropha P22-attB incorporating attB sequence

Carrying out PCR amplification by taking Ralstonia eutropha H16 genome as a template to obtain homologous fragments H1 and H2, carrying out PCR amplification by taking plasmid pK18mobsacB as a template to obtain a vector fragment, carrying out PCR amplification by taking a synthetic fragment 04 as a template to obtain a fragment containing an attB sequence corresponding to P22, and carrying out PCR amplification according to a commercial kit (Gibson)

Master Mix, purchased from New England Biolabs (NEB) Inc.) ligated H1, H2 and attB sequences to the vector fragment by the Gibson Assembly method to give recombinant plasmid pK18 mobsacB-P22. The primers used are as follows:

the sequence of synthetic fragment 04 is:

GGCAGAGAGACAATCAAATCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCCAGGAAACAGCTATGACGGTACGACCTTCGCATTACGAATGCGCTGCACTGGCCGTCGTTTTACAACCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGCCTGCCGGCCTGGTTCAAC(SEQ ID NO:15)

wherein the attB sequence corresponding to P22 is:

ACGACCTTCGCATTACGAATGCGCTGC(SEQ ID NO:16)

the recombinant plasmid pK18mobsacB-P22 is transferred into Escherichia coli S17-1, then transferred into Ralstonia eutropha H16 by a conjugative transformation method, and positive clones are screened by using an LB plate simultaneously containing 200 mug/ml kanamycin and 100 mug/ml apramycin by utilizing the characteristic that the suicide plasmid cannot be replicated in host bacteria. The recombinant plasmid with homologous fragment in the positive clone is integrated into the genome at the specific positions of H1 and H2, and is the first homologous recombinant bacterium.

The first homologous recombinant strain is subjected to single colony culture on an LB plate containing 100mg/ml of sucrose, clones without kanamycin resistance are screened out from the single colonies, and a recombinant strain with the attB sequence of P22 integrated into the genome is identified by PCR (polymerase chain reaction) by using primers primer 7 and primer8, and the obtained recombinant strain is Ralstonia eutropha P22-attB.

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

Carrying out PCR amplification by taking the plasmid pK18mobsacB as a template to obtain a vector fragment, carrying out amplification by taking the synthetic fragment 05 as a template by using a primer to obtain a fragment containing the Bxb1 recombinase gene and the corresponding attP sequence thereof, and carrying out PCR amplification by using a commercial kit (Gibson)

Master Mix, purchased from New England Biolabs (NEB) Inc.) and the fragments were ligated to the vector fragments by the Gibson Assembly method to obtain the recombinant plasmid pBxb 1-attP. The primers used are as follows:

the sequence of synthetic fragment 05 was:

CACACAGGAAACAGCTATGACCTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCTACGACATCCCGGTGTGTAGCCGTTCGACCACGCTGCCGAGCCTGAGATGCTGCTCGTACTCTTGCAGATCCCCGAAGTCGATCGTGCGAGTCAGCCCGCCGCGGACGTCGAACGTCAGCCGAACGTTCATCGACCGAAGCCAGGTGTTCTTTGCCGCGGTGTCCTGCTCCCGCCACCAGTCCCCGAACCGCTGCCCGGTCTCGCGCCACTCCCAGCCAGACGGGCGAGCCTCTAGGCCCTCCAGCTCCTCTTGCCGCGCGGCCAGCGCCGCAATACGGGCATCCAGTGCTTCTCGCTGCGGAGAGCCGGCCCGGTAGGCCGGGGAGCCGATCAGCGACGTCAGGTCCACCAGCTCCGCGTTCACCTCCGCGAGTTCGACCGCGGAGTCCGAGCCGGCTACCCAGACTTTCTCCAGACGCTCCGCGTCCCCGAGCAGATCCAGCACCTGCTCCTCGCAGAACGCGTCCCACTCGGCCATCGCCACCGTGCCGTTCCCGCAGTGCTTCGGGAACCCCATCGAGCGGCAGCGGTAGCGCGGGTGCTTACGTCCTCCCCCGGCGAACTTGTACGCGGGCTCCCCGCACACCGCGCAGAACAACACCCGCAGCAGCAGCGACGGGGTAGACACCGCGGGCTTCGCCCGGGAGGTCTTCACGAGCTCGGCGCGCAGCGCCTCCAGCTGCTCACGGGTCAGGATCGGCTCAGCCCGCACCAGCGGGGCTCCGTCGTCGTCTCGGACGGTCTTACCGTTCAGAGTCGCGTACCCGAGCATCGCCTCGGAGATCATCGATCGCTTCAGCGCGGTAGCCGACCACTCCCGGCCCTGCGGCTCGCGGCCTTGCAGCTGCGCGAAGTAGTCCTTCGGCGACAGGACACCACGCCGGTTCAGGTCGTGGGCCACCAGGTGCAGCGGCTCGTGGTTGTCGACGACGCGGTGATACACCTCGAGGATGCGCTCTCGCTGCACAGGGTCCGGCACCAGCCGCCACTCCCCGTCCACGCGCGTAGGCAGGTATCCCCACGGCGGCAGGGATCCTCGGTATTTCCCGGCGCGGATATTGAAATGCGCAGCCGAACGGTTCCGCTCTTTGATCGCTTCTAATTCCATCTGCGCCACCGTTCCCATAAGCGCGATGACGACCGCCGCAAACGGCGTCGTCGTATCGAAGTGCGCTTCGGTCGCGGAGACGACCAGCTTCTTGTGGTCCTCGGCCCAGTGGACCAGCTGTTGCAGATGCCGGATCGATCGGGTCAACCGGTCTACCCGGTACGCCACGATCACGTCGAACGGTTGCTCCTCGAACGCTAGCCACCGGGCCAGGTTCGGTCTGCGCTTCCGGTCGAACGGATCGACCGCCCCGGAGACGTCCAGATCCTCCGCTACCCCGACGACGTCCCAGCCGCGCTGGGCGCAGAGCTGCTGGCAAGACTCCAGCTGACGCTCCGGTGAAGTCGTAGCATCGGTGACGCGGGACAGGCGGATGACTACCAGGGCTCTCATCTAGTATTTCTCCTCTTTCTCTAGTATTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCGCAGGTCAAAGGGTATACTGGGATTCCAGTGAACGCAAGGGTTTGTACCGTACACCACTGAGACCGCGGTGGTTGACCAGACAAACCACGAACTGGCCGTCGTTTTACAAC(SEQ ID NO:19)

wherein the gene sequence of Bxb1 recombinase is as follows:

ATGAGAGCCCTGGTAGTCATCCGCCTGTCCCGCGTCACCGATGCTACGACTTCACCGGAGCGTCAGCTGGAGTCTTGCCAGCAGCTCTGCGCCCAGCGCGGCTGGGACGTCGTCGGGGTAGCGGAGGATCTGGACGTCTCCGGGGCGGTCGATCCGTTCGACCGGAAGCGCAGACCGAACCTGGCCCGGTGGCTAGCGTTCGAGGAGCAACCGTTCGACGTGATCGTGGCGTACCGGGTAGACCGGTTGACCCGATCGATCCGGCATCTGCAACAGCTGGTCCACTGGGCCGAGGACCACAAGAAGCTGGTCGTCTCCGCGACCGAAGCGCACTTCGATACGACGACGCCGTTTGCGGCGGTCGTCATCGCGCTTATGGGAACGGTGGCGCAGATGGAATTAGAAGCGATCAAAGAGCGGAACCGTTCGGCTGCGCATTTCAATATCCGCGCCGGGAAATACCGAGGATCCCTGCCGCCGTGGGGATACCTGCCTACGCGCGTGGACGGGGAGTGGCGGCTGGTGCCGGACCCTGTGCAGCGAGAGCGCATCCTCGAGGTGTATCACCGCGTCGTCGACAACCACGAGCCGCTGCACCTGGTGGCCCACGACCTGAACCGGCGTGGTGTCCTGTCGCCGAAGGACTACTTCGCGCAGCTGCAAGGCCGCGAGCCGCAGGGCCGGGAGTGGTCGGCTACCGCGCTGAAGCGATCGATGATCTCCGAGGCGATGCTCGGGTACGCGACTCTGAACGGTAAGACCGTCCGAGACGACGACGGAGCCCCGCTGGTGCGGGCTGAGCCGATCCTGACCCGTGAGCAGCTGGAGGCGCTGCGCGCCGAGCTCGTGAAGACCTCCCGGGCGAAGCCCGCGGTGTCTACCCCGTCGCTGCTGCTGCGGGTGTTGTTCTGCGCGGTGTGCGGGGAGCCCGCGTACAAGTTCGCCGGGGGAGGACGTAAGCACCCGCGCTACCGCTGCCGCTCGATGGGGTTCCCGAAGCACTGCGGGAACGGCACGGTGGCGATGGCCGAGTGGGACGCGTTCTGCGAGGAGCAGGTGCTGGATCTGCTCGGGGACGCGGAGCGTCTGGAGAAAGTCTGGGTAGCCGGCTCGGACTCCGCGGTCGAACTCGCGGAGGTGAACGCGGAGCTGGTGGACCTGACGTCGCTGATCGGCTCCCCGGCCTACCGGGCCGGCTCTCCGCAGCGAGAAGCACTGGATGCCCGTATTGCGGCGCTGGCCGCGCGGCAAGAGGAGCTGGAGGGCCTAGAGGCTCGCCCGTCTGGCTGGGAGTGGCGCGAGACCGGGCAGCGGTTCGGGGACTGGTGGCGGGAGCAGGACACCGCGGCAAAGAACACCTGGCTTCGGTCGATGAACGTTCGGCTGACGTTCGACGTCCGCGGCGGGCTGACTCGCACGATCGACTTCGGGGATCTGCAAGAGTACGAGCAGCATCTCAGGCTCGGCAGCGTGGTCGAACGGCTACACACCGGGATGTCGTAG(SEQ ID NO:20)

the amino acid sequence of the Bxb1 recombinase is as follows:

MRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDLDVSGAVDPFDRKRRPNLARWLAFEEQPFDVIVAYRVDRLTRSIRHLQQLVHWAEDHKKLVVSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWGYLPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQGREPQGREWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRAELVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGTVAMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAVELAEVNAELVDLTSLIGSPAYRAGSPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRSMNVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS(SEQ ID NO:21)

the corresponding attP sequence of Bxb1 is:

TCGTGGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAACCC (SEQ ID NO:22) comparative example 4: construction of a recombinant plasmid pPhiC31-attP containing the recombinase and the corresponding attP sequence

Carrying out PCR amplification by taking the plasmid pK18mobsacB as a template to obtain a vector fragment, carrying out amplification by taking the synthetic fragment 06 as a template by using a primer to obtain a fragment containing the PhiC31 recombinase gene and the corresponding attP sequence thereof, and carrying out PCR amplification by using a commercial kit (Gibson)

Master Mix, purchased from New England Biolabs (NEB) Inc.) and the fragments were ligated to the vector fragments by the Gibson Assembly method to obtain the recombinant plasmid pPhiC 31-attP. The primers used are as follows:

the sequence of synthetic fragment 06 was:

CACACAGGAAACAGCTATGACCTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCTACGCCGCTACGTCTTCCGTGCCGTCCTGGGCGTCGTCTTCGTCGTCGTCGGTCGGCGGCTTCGCCCACGTGATCGAAGCGCGCTTCTCGATGGGCGTTCCCTGCCCCCTGCCCGTAGTCGACTTCGTGACAACGATCTTGTCTACGAAGAGCCCGACGAACACGCGCTTGTCGTCTACTGACGCGCGCCCCCACCACGACTTAGGGCCGGTCGGGTCAGCGTCGGCGTCTTCGGGGAACCATTGGTCAAGGGGAAGCTTCGGGGCTTCGGCGGCTTCAAGTTCGGCAAGCCGCTCTTCCGCCCCTTGCTGCCGGAGCGTCAGCGCTGCCTGTTGCTTCCGGAAGTGCTTCCTGCCAACGGGTCCGTCGTACGCGCCTGCCGCGCGGTCTTCGTACAGCTCTTCAAGGGCGTTCAGGGCGTCGGCGCGCTCCGCAACAAGGTTCGCCCGTTCGCCGCTCTTCTCAGGCGCCTCAGTGAGCTTGCCGAAGCGTCGGGCGGCTTCCCACAGAAGCGCCAACGTCTCTTCGTCGCCTTCGGCGTGCCTGATCTTGTTGAAGATGCGTTCCGCAACGAACTTGTCGAGTGCCGCCATGCTGACGTTGCACGTGCCTTCGTGCTGCCCAGGTGCGGACGGGTCGACCACCTTCCGGCGACGGCAGCGGTAAGAGTCCTTGATCGATTCTTCCCCGCGCTTCGAAGTCATGACGGCGCCACACTCGCAGTACAGCTTGTCCATGGCGGACAGAATGGCTTGCCCCCGGGAAAGCCCCTTGCCGCGCCCCCTGCCGTCCAACCACGCCTGAAGCTCATACCACTCAGCGGGCTCGATGATCGGTCCGCAATCAAGCTCGACCGGCCGGAGCGTGATCGGGTCGCGCTGAATGCGGTAACCCTCAATCTTCGTGGTCGGCGTGCCGTCCGGCTTCTTCTTGTAGATCACCTCAGCGGCGAAGCCCGCAATACGCGGGTCCCGAAGGATTCGCATAACGGTTGCCGGGTCCCAGGCGCTTGAAGCGGTCTTCTTCCCAATCGTCTCGCCCCGGGTCGGCACGGCGTCAGCGTCCATGCGCTTACAAAGCCCCGTGATGCTGCCCGGGTGAATGGCGGCTTGACTGCCCGGCTTGAAGGGAAGGTGTTTGTGCGTCTTGATCTCACGCCACCACCACCGGATTACGTCGGGCTCGAACTCGAAGGGTCCGGTAAGGGGAGTGGTCGAGTGCGCAAGCTTGTTGATGACGACATTGACCATTCGGCCGTTGCGCGTGATCTCCTTCGTCTCCGAAACAAGCTCGAAGCCGTAAGGCGCCTTCCCGCCGACGTACCCGCCCAATTCGCGCTGAAGGTTCTTCGTGTCGAGAATCTTCGCCGACTTCAGCGAAGATTCTTTGTGCGACGCGTCGAGCCGCATAATCAGGTGAATCAGGTCCATGACGTTTCCCTGCCGGAAGACGCCTTCCTGAGTGGAAACAATCGTCACGCCCAGGGCGAGCAATTCCGAGACAATCGGAATCGCGTCCATGACCTTCAGGCGCGAGAAGCGCGACACGTCATAGACAATGATCATGTTGAGCCGCCCGGCGCGGCATTCGTTCAGGATGCGTTCGAACTCCGGGCGCTCCGCCGTCCCGAACGCCGACGTGCCCGGCGCTTCGCTGAAATGCCCGACGAACCTGAACCGGCCCCCGTCGCGCTCGACTTCGCGCTGAAGGTCGGCCGCCTTGTCTTCGTTGGCGCTACGCTGTGTCGCTGGGCTTGCTGCGCTCGAATTCTCGCGCTCGCGCGACTGACGGTCGTAAGCACCCGCGTACGTGTCCATCTAGTATTTCTCCTCTTTCTCTAGTATTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCGCAGGTCAAAGGGTATACTGGGATTCCAGTGAACGCAACCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGACTGGCCGTCGTTTTACAAC(SEQ ID NO:23)

wherein the gene sequence of the PhiC31 recombinase is as follows:

ATGGACACGTACGCGGGTGCTTACGACCGTCAGTCGCGCGAGCGCGAGAATTCGAGCGCAGCAAGCCCAGCGACACAGCGTAGCGCCAACGAAGACAAGGCGGCCGACCTTCAGCGCGAAGTCGAGCGCGACGGGGGCCGGTTCAGGTTCGTCGGGCATTTCAGCGAAGCGCCGGGCACGTCGGCGTTCGGGACGGCGGAGCGCCCGGAGTTCGAACGCATCCTGAACGAATGCCGCGCCGGGCGGCTCAACATGATCATTGTCTATGACGTGTCGCGCTTCTCGCGCCTGAAGGTCATGGACGCGATTCCGATTGTCTCGGAATTGCTCGCCCTGGGCGTGACGATTGTTTCCACTCAGGAAGGCGTCTTCCGGCAGGGAAACGTCATGGACCTGATTCACCTGATTATGCGGCTCGACGCGTCGCACAAAGAATCTTCGCTGAAGTCGGCGAAGATTCTCGACACGAAGAACCTTCAGCGCGAATTGGGCGGGTACGTCGGCGGGAAGGCGCCTTACGGCTTCGAGCTTGTTTCGGAGACGAAGGAGATCACGCGCAACGGCCGAATGGTCAATGTCGTCATCAACAAGCTTGCGCACTCGACCACTCCCCTTACCGGACCCTTCGAGTTCGAGCCCGACGTAATCCGGTGGTGGTGGCGTGAGATCAAGACGCACAAACACCTTCCCTTCAAGCCGGGCAGTCAAGCCGCCATTCACCCGGGCAGCATCACGGGGCTTTGTAAGCGCATGGACGCTGACGCCGTGCCGACCCGGGGCGAGACGATTGGGAAGAAGACCGCTTCAAGCGCCTGGGACCCGGCAACCGTTATGCGAATCCTTCGGGACCCGCGTATTGCGGGCTTCGCCGCTGAGGTGATCTACAAGAAGAAGCCGGACGGCACGCCGACCACGAAGATTGAGGGTTACCGCATTCAGCGCGACCCGATCACGCTCCGGCCGGTCGAGCTTGATTGCGGACCGATCATCGAGCCCGCTGAGTGGTATGAGCTTCAGGCGTGGTTGGACGGCAGGGGGCGCGGCAAGGGGCTTTCCCGGGGGCAAGCCATTCTGTCCGCCATGGACAAGCTGTACTGCGAGTGTGGCGCCGTCATGACTTCGAAGCGCGGGGAAGAATCGATCAAGGACTCTTACCGCTGCCGTCGCCGGAAGGTGGTCGACCCGTCCGCACCTGGGCAGCACGAAGGCACGTGCAACGTCAGCATGGCGGCACTCGACAAGTTCGTTGCGGAACGCATCTTCAACAAGATCAGGCACGCCGAAGGCGACGAAGAGACGTTGGCGCTTCTGTGGGAAGCCGCCCGACGCTTCGGCAAGCTCACTGAGGCGCCTGAGAAGAGCGGCGAACGGGCGAACCTTGTTGCGGAGCGCGCCGACGCCCTGAACGCCCTTGAAGAGCTGTACGAAGACCGCGCGGCAGGCGCGTACGACGGACCCGTTGGCAGGAAGCACTTCCGGAAGCAACAGGCAGCGCTGACGCTCCGGCAGCAAGGGGCGGAAGAGCGGCTTGCCGAACTTGAAGCCGCCGAAGCCCCGAAGCTTCCCCTTGACCAATGGTTCCCCGAAGACGCCGACGCTGACCCGACCGGCCCTAAGTCGTGGTGGGGGCGCGCGTCAGTAGACGACAAGCGCGTGTTCGTCGGGCTCTTCGTAGACAAGATCGTTGTCACGAAGTCGACTACGGGCAGGGGGCAGGGAACGCCCATCGAGAAGCGCGCTTCGATCACGTGGGCGAAGCCGCCGACCGACGACGACGAAGACGACGCCCAGGACGGCACGGAAGACGTAGCGGCGTAG(SEQ ID NO:24)

the amino acid sequence of the PhiC31 recombinase is as follows:

MDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDGGRFRFVGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIPIVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQRELGGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWREIKTHKHLPFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRILRDPRIAGFAAEVIYKKKPDGTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQAWLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPGQHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERANLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAEAPKLPLDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPIEKRASITWAKPPTDDDEDDAQDGTEDVAA(SEQ ID NO:25)

the corresponding attP sequence of PhiC31 is:

CCCCCAACTGAGAGAACTCAAAGGTTACCCCAGTTGGGG(SEQ ID NO:26)

comparative example 5: construction of recombinant plasmid pTP901-attP containing recombinase and corresponding attP sequence

Carrying out PCR amplification by taking plasmid pK18mobsacB as a template to obtain a vector fragment, carrying out amplification by taking a synthetic fragment 07 as a template by using a primer to obtain a fragment containing a TP901 recombinase gene and an attP sequence corresponding to the TP recombinase gene, and carrying out PCR amplification by using a commercial kit (Gibson)

Master Mix, purchased from New England Biolabs (NEB) Inc.) and the fragments were ligated to the vector fragment by the Gibson Assembly method to obtain the recombinant plasmid pTP 901-attP. The primers used are as follows:

the sequence of synthetic fragment 07 is:

CACACAGGAAACAGCTATGACCTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGTTAAGCAGCCAGAGCGTAGTTTTCGTCCTTAGCAGCACCGGTAGCGAGTTGGAATTTAAATATGATATCTACATTATCAGCAGTAACATCAACCTTTGATACAAGGTTGTTGACGATTTTCTTTTTATTATCATATGATAGTTCATTAATCGGAATTGAGCCCAACTGAGTTTTAACTAACTCAAAAACATCAGTAGAGTCATTAAATTTATTTTCGCTAATCTTAGCTTTAAGCAGCTTTTTCTCAGCCTGAAGGGAATCAGTACGATCTTTCAACTCATCCATAGTGATAAAATCATTTAGGTACAAATCAGAGTTCTTTTGTATTTTTTTATCGATCTGTGAAATTTGCTTTTTAAATGACGAAGTATCAAGAATAGGTTGGTTGTTGCCATTGATAATTTTCAATAAGGAGTCATTATTTTCTTGAAATCCAATCAGGTTGTCAATAACAGTATTTTCTAAATTACTTAAATCATAAGTTCCTGAATCACACTTTTTATTGTCATTATATACTGTAATTCCTTTTGTTTTTCGAGGAAATCTATTTGCACAGTGATATTTCATAGTGCGGCTTCCATCTTTTCTTTTGTGGCCAAGAACAATTTTTAAAGGTGCTCCACAGTAACCGCACCTTGCCATCCCTGACAGCATATATTTAGCTTGGAAAGGTCTAGGGTTGTTATTTCTTTCATAAGTCTGCTGTTGTCTTTCTTCTAGCTCTTTTTGAACTTTTAAATAAGTCTCATAAGGGATAATTGGTTTGTGCATACCTTCAAATAGGCTGTCCTTAAATTTGATATAACCACAGTAAACTGGATTATCAAGTGTTTGTCTTAGGGTACGATAAGACCACGGTATATCTTTACCGATGTGTCCAGATTCATTGAGTTTATCTCTTAATTTTGTAAGTGATATTCCTGATAAATAATCAGTGAATATTTGTTCAACTATTGTAGCTTGTAAAGGAACAATTTCTAATATACCTGTCTTTCTGTTGTGGTAATACCCAAAAGCTGTCTTAGTCCACATCATAGACTTACCAGATTTCGCTCGCCCTAGTTTACCCATAGTCATGCGTTCTTTTATATTCTCTCTTTCAAACTCATTAATTGCAGAAAGAATAGTGAGAAACAAGCTACCCATAGCAGAAGAAGTATCAATACTTTCATTAAGCGAGATAAAGTCTATTTTATTTTTTGTGAACACATCCTTAACAAGATAAAGAGTATCTCTTACACTACGTGAAAGGCGGTCTAGCTTATATACAAGAACTGTATCAAAAGCTTTATTCTCGATATCGTTGATTAATCTTTGCATTGCTGGGCGTTCAAGTTTGGCCCCTGAAAAACCAGCATCAGTATAAGTATCAGATACTTGCCACCCCATTGCTTCAGCATATTTTGTTAAACGGTCAATTTGCTCATCAATTGAGAAGCCTTCCTCTGCTTGGTTAGTAGTGGATACTCGTGTATAGATTGCTACTTTCTTAGTGCCGGCCTGGTGGTGATGGTGATGATGTTTCATCTAGTATTTCTCCTCTTTCTCTAGTATTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCGCAGGTCAAAGGGTATACTGGGATTCCAGTGAACGCAAAAAAGGAGTTTTTTAGTTACCTTAATTGAAATAAACGAAATAAAAACTCGACTGGCCGTCGTTTTACAAC(SEQ ID NO:27)

wherein the gene sequence of the TP901 recombinase is as follows:

ATGAAACATCATCACCATCACCACCAGGCCGGCACTAAGAAAGTAGCAATCTATACACGAGTATCCACTACTAACCAAGCAGAGGAAGGCTTCTCAATTGATGAGCAAATTGACCGTTTAACAAAATATGCTGAAGCAATGGGGTGGCAAGTATCTGATACTTATACTGATGCTGGTTTTTCAGGGGCCAAACTTGAACGCCCAGCAATGCAAAGATTAATCAACGATATCGAGAATAAAGCTTTTGATACAGTTCTTGTATATAAGCTAGACCGCCTTTCACGTAGTGTAAGAGATACTCTTTATCTTGTTAAGGATGTGTTCACAAAAAATAAAATAGACTTTATCTCGCTTAATGAAAGTATTGATACTTCTTCTGCTATGGGTAGCTTGTTTCTCACTATTCTTTCTGCAATTAATGAGTTTGAAAGAGAGAATATAAAAGAACGCATGACTATGGGTAAACTAGGGCGAGCGAAATCTGGTAAGTCTATGATGTGGACTAAGACAGCTTTTGGGTATTACCACAACAGAAAGACAGGTATATTAGAAATTGTTCCTTTACAAGCTACAATAGTTGAACAAATATTCACTGATTATTTATCAGGAATATCACTTACAAAATTAAGAGATAAACTCAATGAATCTGGACACATCGGTAAAGATATACCGTGGTCTTATCGTACCCTAAGACAAACACTTGATAATCCAGTTTACTGTGGTTATATCAAATTTAAGGACAGCCTATTTGAAGGTATGCACAAACCAATTATCCCTTATGAGACTTATTTAAAAGTTCAAAAAGAGCTAGAAGAAAGACAACAGCAGACTTATGAAAGAAATAACAACCCTAGACCTTTCCAAGCTAAATATATGCTGTCAGGGATGGCAAGGTGCGGTTACTGTGGAGCACCTTTAAAAATTGTTCTTGGCCACAAAAGAAAAGATGGAAGCCGCACTATGAAATATCACTGTGCAAATAGATTTCCTCGAAAAACAAAAGGAATTACAGTATATAATGACAATAAAAAGTGTGATTCAGGAACTTATGATTTAAGTAATTTAGAAAATACTGTTATTGACAACCTGATTGGATTTCAAGAAAATAATGACTCCTTATTGAAAATTATCAATGGCAACAACCAACCTATTCTTGATACTTCGTCATTTAAAAAGCAAATTTCACAGATCGATAAAAAAATACAAAAGAACTCTGATTTGTACCTAAATGATTTTATCACTATGGATGAGTTGAAAGATCGTACTGATTCCCTTCAGGCTGAGAAAAAGCTGCTTAAAGCTAAGATTAGCGAAAATAAATTTAATGACTCTACTGATGTTTTTGAGTTAGTTAAAACTCAGTTGGGCTCAATTCCGATTAATGAACTATCATATGATAATAAAAAGAAAATCGTCAACAACCTTGTATCAAAGGTTGATGTTACTGCTGATAATGTAGATATCATATTTAAATTCCAACTCGCTACCGGTGCTGCTAAGGACGAAAACTACGCTCTGGCTGCTTAA(SEQ ID NO:28)

the amino acid sequence of the TP901 recombinase is as follows:

MKHHHHHHQAGTKKVAIYTRVSTTNQAEEGFSIDEQIDRLTKYAEAMGWQVSDTYTDAGFSGAKLERPAMQRLINDIENKAFDTVLVYKLDRLSRSVRDTLYLVKDVFTKNKIDFISLNESIDTSSAMGSLFLTILSAINEFERENIKERMTMGKLGRAKSGKSMMWTKTAFGYYHNRKTGILEIVPLQATIVEQIFTDYLSGISLTKLRDKLNESGHIGKDIPWSYRTLRQTLDNPVYCGYIKFKDSLFEGMHKPIIPYETYLKVQKELEERQQQTYERNNNPRPFQAKYMLSGMARCGYCGAPLKIVLGHKRKDGSRTMKYHCANRFPRKTKGITVYNDNKKCDSGTYDLSNLENTVIDNLIGFQENNDSLLKIINGNNQPILDTSSFKKQISQIDKKIQKNSDLYLNDFITMDELKDRTDSLQAEKKLLKAKISENKFNDSTDVFELVKTQLGSIPINELSYDNKKKIVNNLVSKVDVTADNVDIIFKFQLATGAAKDENYALAA(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 the recombinase and the corresponding attP sequence

Similarly, a vector fragment was obtained by PCR amplification using plasmid pK18mobsacB as a template, a fragment containing the P22 recombinase gene and its corresponding attP sequence was obtained by amplification using primers and synthetic fragment 08 as a template, according to a commercial kit (Gibson)

Master Mix, purchased from New England Biolabs (NEB) Inc.) and the fragments were ligated to the vector fragments by the Gibson Assembly method to obtain the recombinant plasmid pP 22-attP. Make itThe primers used are as follows:

the sequence of synthetic fragment 08 is:

CACACAGGAAACAGCTATGACCTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCTACGTATTATTCGTGCCTTCCTTATTTTTACTGTGGGACATATTTGGGACAGAAGTACCAAAAATCGAGTCAATTTGTCGAGCATGTTCAGTCAGGTGATTTGGTGCCAGATGAGCATATCGGCGAACCATTTCGATAGACTCCCAGCCACCCATTTCCTGCAATACCGAAATCGGAACGCCAGCCTGAACTAACCAACTTGCCCACGTGTGCCTCAGGTCATGAAAACGGAAGTCTTCAATGCCCGCTCGTTTTAATGCTGCCCTCCATGCAGTATTAGCGTCATAGCGCATCTTCCTCACTACAGGTGATTTAGTTCCGTCTGGTTTGGTGCTGCTTTCCTTGTAGACGAACACCCATTTGTGATGATTGCCGATTTGCTTTTTCAGCACCCGGCAAGCGGTATCATTCAGCGCCACTCCAATGGCATGATTAGACTTGCTTTGTTCCGGGTGTATCCATGCCACCTTTCGTTGCATGTCTATCTGCTGCCACTCCAGATTGATAATGTTAGACCGCCTTAAGCCAGTAGAAAGCGCAAACTCTACGACTGACTTTAGCGGTTCCTGGCATTCATCAATCAACCTTTTTGCCTCGTGAGGCTCAAGCCAGCGGATACGCTTATTTTTCGGCTGAGGAACTTTGATGATCGGAGCCTTATCCAGCATCTTCCATTCGCGTTCAGCAGCCCGGAGGAGTGCCTTAATGAATGAAAGGTGAGTTGCTTTTGTAGCTACTGCTGCCGGCTTAGGCTTGAATACCGGAGGCTGCTTCCCATTCTTCCTGCATGCTTCATCCATTAACTTCCAGTTTTCCTCATGCCGCCGATTAGTTATCTTCTGGATGGCGGAGTAAATCTTCGTCTCGGTAATATCCTTCAACTGCATTCCTGCAAAATGCTGGAGCCAGAATCCTATCCGACTCTTGTCATCATCCAGCGACTTCTTATGCGCCTTCTCCTCTAACCACCTGACACAGGCCCCCTCAAAAGTCATGTCAGGCGTCTCTCCTAATTTACTTACCCTCCATGCTTCTGCCTTCAGCTTGTCATGAAGCTCTGTGGCCTGCCTTTTGTCCTTTGTCCCAAGAGACTGCTTAAATCTTTTGCCGTTCGGCAATGTGAAACTGGCGTACCAGGTTTCACCTCTGCGGAATAGTGACATCTAGTATTTCTCCTCTTTCTCTAGTATTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCGCAGGTCAAAGGGTATACTGGGATTCCAGTGAACGCAACTAAGTGGTTTGGGACAAAAATGGGACATACAAATCTTTGCATCGGTTTGCAAGGCTTTGCATGTCTTTCGAAGATGGGACGTGTGAGCGCAGGTATGACGTGGTATGTTGTTGACTTAAAAGGTAGTTCTTATAATTCGTAATGCGAAGGTCGTAGGTTCGACTCCTATTATCGGCACCAGTTAAATCAAATACTTACGTATTATTCGTGCCTTCCTTATTTTTACTGTGGGACATATTTGGGACAGAAGTACCAAAAAACTGGCCGTCGTTTTACAAC(SEQ ID NO:31)

wherein the gene sequence of the P22 recombinase is as follows:

ATGTCACTATTCCGCAGAGGTGAAACCTGGTACGCCAGTTTCACATTGCCGAACGGCAAAAGATTTAAGCAGTCTCTTGGGACAAAGGACAAAAGGCAGGCCACAGAGCTTCATGACAAGCTGAAGGCAGAAGCATGGAGGGTAAGTAAATTAGGAGAGACGCCTGACATGACTTTTGAGGGGGCCTGTGTCAGGTGGTTAGAGGAGAAGGCGCATAAGAAGTCGCTGGATGATGACAAGAGTCGGATAGGATTCTGGCTCCAGCATTTTGCAGGAATGCAGTTGAAGGATATTACCGAGACGAAGATTTACTCCGCCATCCAGAAGATAACTAATCGGCGGCATGAGGAAAACTGGAAGTTAATGGATGAAGCATGCAGGAAGAATGGGAAGCAGCCTCCGGTATTCAAGCCTAAGCCGGCAGCAGTAGCTACAAAAGCAACTCACCTTTCATTCATTAAGGCACTCCTCCGGGCTGCTGAACGCGAATGGAAGATGCTGGATAAGGCTCCGATCATCAAAGTTCCTCAGCCGAAAAATAAGCGTATCCGCTGGCTTGAGCCTCACGAGGCAAAAAGGTTGATTGATGAATGCCAGGAACCGCTAAAGTCAGTCGTAGAGTTTGCGCTTTCTACTGGCTTAAGGCGGTCTAACATTATCAATCTGGAGTGGCAGCAGATAGACATGCAACGAAAGGTGGCATGGATACACCCGGAACAAAGCAAGTCTAATCATGCCATTGGAGTGGCGCTGAATGATACCGCTTGCCGGGTGCTGAAAAAGCAAATCGGCAATCATCACAAATGGGTGTTCGTCTACAAGGAAAGCAGCACCAAACCAGACGGAACTAAATCACCTGTAGTGAGGAAGATGCGCTATGACGCTAATACTGCATGGAGGGCAGCATTAAAACGAGCGGGCATTGAAGACTTCCGTTTTCATGACCTGAGGCACACGTGGGCAAGTTGGTTAGTTCAGGCTGGCGTTCCGATTTCGGTATTGCAGGAAATGGGTGGCTGGGAGTCTATCGAAATGGTTCGCCGATATGCTCATCTGGCACCAAATCACCTGACTGAACATGCTCGACAAATTGACTCGATTTTTGGTACTTCTGTCCCAAATATGTCCCACAGTAAAAATAAGGAAGGCACGAATAATACGTAG(SEQ ID NO:32)

the amino acid sequence of the P22 recombinase is:

MSLFRRGETWYASFTLPNGKRFKQSLGTKDKRQATELHDKLKAEAWRVSKLGETPDMTFEGACVRWLEEKAHKKSLDDDKSRIGFWLQHFAGMQLKDITETKIYSAIQKITNRRHEENWKLMDEACRKNGKQPPVFKPKPAAVATKATHLSFIKALLRAAEREWKMLDKAPIIKVPQPKNKRIRWLEPHEAKRLIDECQEPLKSVVEFALSTGLRRSNIINLEWQQIDMQRKVAWIHPEQSKSNHAIGVALNDTACRVLKKQIGNHHKWVFVYKESSTKPDGTKSPVVRKMRYDANTAWRAALKRAGIEDFRFHDLRHTWASWLVQAGVPISVLQEMGGWESIEMVRRYAHLAPNHLTEHARQIDSIFGTSVPNMSHSKNKEGTNNT(SEQ ID NO:33)

the attP sequence for P22 is:

TTTTTGGTACTTCTGTCCCAAATATGTCCCACAGTAAAAATAAGGAAGGCACGAATAATACGTAAGTATTTGATTTAACTGGTGCCGATAATAGGAGTCGAACCTACGACCTTCGCATTACGAATTATAAGAACTACCTTTTAAGTCAACAACATACCACGTCATACCTGCGCTCACACGTCCCATCTTCGAAAGACATGCAAAGCCTTGCAAACCGATGCAAAGATTTGTATGTCCCATTTTTGTCCCAAACCACTTAG(SEQ ID NO:34)

example 3: recombinase-mediated attB-attP recombination

The recombinant plasmid pBxb1-attP is transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha Bxb1-attB by a conjugative transformation method, and by utilizing the characteristic that suicide plasmid can not be replicated in host bacteria, an LB plate sieve containing 200 mug/ml kanamycin and 100 mug/ml apramycin is used. If recombinase Bxb1 is functional, recombination between attB and attP sequences will be mediated, resulting in integration of plasmid pBxb1-attP into the genome.

Comparative example 7: recombinase-mediated attB-attP recombination

The recombinant plasmid pPhiC31-attP is transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha PhiC31-attB by a conjugative transformation method, and an LB plate sieve containing 200 mug/ml kanamycin and 100 mug/ml apramycin is used by utilizing the characteristic that the suicide plasmid cannot replicate in host bacteria. If the recombinase PhiC31 is functional, recombination between attB and attP sequences will be mediated, thereby integrating the plasmid pPhiC1-attP into the genome.

Comparative example 8: recombinase-mediated attB-attP recombination

The recombinant plasmid pTP901-attP is transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha TP901-attB by a conjugation transformation method, and LB plate sieve containing 200 mug/ml kanamycin and 100 mug/ml apramycin is used by utilizing the characteristic that suicide plasmid can not be replicated in host bacteria. If recombinase TP901 is functional, recombination between attB and attP sequences will be mediated, resulting in integration of plasmid pTP901-attP into the genome.

Comparative example 9: recombinase-mediated attB-attP recombination

The recombinant plasmid pP22-attP is transferred into Escherichia coli S17-1, and then transferred into Ralstonia eutropha P22-attB by a conjugative transformation method, and an LB plate sieve containing 200 mug/ml kanamycin and 100 mug/ml apramycin at the same time is used by utilizing the characteristic that the suicide plasmid cannot replicate in host bacteria. If recombinase P22 is functional, recombination between attB and attP sequences will be mediated, resulting in integration of plasmid pP22-attP into the genome.

Experimental example 1: verification of vector integration into genome

After the plasmids of 4 recombinases were transferred to the corresponding Ralstonia eutropha, 8 clones were randomly picked up and verified by PCR using primers gcgcatggcgtctccatg (SEQ ID NO:35) and gtggaccagctgttgcag (SEQ ID NO:36), and the results are shown in the following table:

the above table shows that recombination mediated by the recombinase Bxb1 with attB and attP all gave the expected 862bp band, demonstrating integration of plasmid pBxb1-attP into the genome (FIG. 3), whereas recombination mediated by the recombinases PhiC31, TP901 and P22 with attB and attP was unsuccessful.

It follows that it is not necessary or foreseeable that a recombinase that has proven useful in some host microorganisms will function in another microorganism. Only Bxb1 in 4 recombinant enzymes tested by the invention has the function in Ralstonia eutropha, and can be applied to the integration of exogenous sequences; however, none of the other 3 recombinases had a function and were not suitable for use in Ralstonia eutropha strains.

Example 4: deletion of vector backbone with helper plasmid

The above examples demonstrate that recombinase Bxb1 has function in Ralstonia eutropha, and DNA fragments to be integrated can be integrated on a vector containing Bxb1 gene and corresponding attP sequence together with the vector to the genome of recombinant bacteria Ralstonia eutropha Bxb 1-attB. In practice, however, it is more preferable that the vector portion other than the DNA fragment to be integrated be deleted. This example uses another set of recombinases to achieve this goal.

In the form of 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, cloning differential anti-genetic cassettes. Gene.166,175-176) as template for PCR amplification to obtain replicon fragments; amplifying by using a primer and taking the synthesized segment 09 as a template to obtain a DNA segment, wherein the segment contains a VCre recombinase gene, a kanamycin resistance gene and a spectinomycin resistance gene; according to commercial kits (Gibson)

Master Mix, purchased from New England Biolabs (NEB) Inc.) and the fragments were ligated to the replicon fragments by the Gibson Assembly method to obtain recombinant plasmid pVCre. The primers used are as follows:

the sequence of synthetic fragment 09 was:

GAGCCAGCCGGTGGCCGCCTACATGGCTCTGCTGTAGTTCACCCTTGGCGTCCAACCAGCGGCACCAGCGGCGCCTGAGAGGGGCGCGCCCAGCTGTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTTTAATTAAAGCGGATAACAATTTCACACAGGACAACTGAGACCGGAATTGGTCTCAACGTACGTCTCATTTTCGCCAGATATCGACGTCTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCTAGACATCATTAATTCCTAATTTTTGTTGACACTCTATCGTTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGAGAAAAGAATTCAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAATACTAGAGAAAGAGGAGAAATACTAGATGATCGAGAACCAGCTGAGCCTGCTGGGTGATTTCAGCGGCGTGCGTCCGGACGATGTTAAGACCGCGATCCAGGCGGCGCAAAAGAAAGGTATTAACGTTGCGGAGAACGAACAATTCAAAGCGGCGTTTGAGCACCTGCTGAACGAGTTCAAGAAACGTGAGGAACGTTACAGCCCGAACACCCTGCGTCGTCTGGAAAGCGCGTGGACCTGCTTTGTGGATTGGTGCCTGGCGAACCATCGTCACAGCCTGCCGGCGACCCCGGACACCGTTGAGGCGTTCTTTATCGAACGTGCGGAGGAACTGCACCGTAACACCCTGAGCGTGTACCGTTGGGCGATTAGCCGTGTTCATCGTGTTGCGGGTTGCCCGGACCCGTGCCTGGATATCTATGTGGAGGATCGTCTGAAGGCGATTGCGCGTAAGAAAGTGCGTGAGGGCGAAGCGGTTAAACAGGCGAGCCCGTTTAACGAACAACACCTGCTGAAGCTGACCAGCCTGTGGTACCGTAGCGACAAACTGCTGCTGCGTCGTAACCTGGCGCTGCTGGCGGTGGCGTATGAGAGCATGCTGCGTGCGAGCGAACTGGCGAACATCCGTGTTAGCGACATGGAGCTGGCGGGTGATGGCACCGCGATTCTGACCATCCCGATTACCAAGACCAACCACAGCGGCGAGCCGGACACCTGCATTCTGAGCCAGGATGTGGTTAGCCTGCTGATGGACTACACCGAAGCGGGCAAGCTGGACATGAGCAGCGATGGTTTCCTGTTTGTGGGCGTTAGCAAACACAACACCTGCATCAAGCCGAAGAAAGATAAACAGACCGGTGAAGTTCTGCACAAGCCGATTACCACCAAAACCGTGGAGGGCGTTTTCTATAGCGCGTGGGAAACCCTGGATCTGGGTCGTCAAGGCGTGAAGCCGTTTACCGCGCACAGCGCGCGTGTTGGTGCGGCGCAGGACCTGCTGAAGAAAGGCTACAACACCCTGCAAATCCAGCAAAGCGGTCGTTGGAGCAGCGGCGCGATGGTTGCGCGTTATGGTCGTGCGATCCTGGCGCGTGACGGCGCGATGGCGCACAGCCGTGTGAAAACCCGTAGCGCGCCGATGCAATGGGGCAAGGACGAGAAAGATTAATGATAAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGAGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTACTAGAGGTCATGCTTGCCATCTGTTTTCTTGCAAGATTACTAGTAGCGGCCGCTGCAGGTCGTGACTGGGAAAACCCTGGCGACTAGTCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAGACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCTATTCTGACCTTGCCATCACGACTGTGCTGGTCATTAAACGCGTATTCAGGCTGACCCTGCGCGCTGCGCAGGGCTTTATTGATTCCATTTTTACACTGATGAATGTTCCGTTGCGCTGCCCGGATTACAGCCGGATCCTCTAGAGTCGACCTGCAGGCATGCTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAAACTAGTAAATAATAAAAAAGCCGGATTAATAATCTGGCTTTTTATATTCTCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTTTTTCGGTATATCCATCCTTTTTCGCACGATATACAGGATTTTGCCAAAGGGTTCGTGTAGACTTTCCTTGGTGTATCCAACGGCGTCAGCCGGGCAGGATAGGTGAAGTAGGCCCACCCGCGAGCGGGTGTTCCTTCTTCACTGTCCCTTATTCGCACCTGGCGGTGCTCAACGGGAATCCTGCTCTGCGAGGCTGGCCGTAGGCCGGCCGCGATGCAGGTGGCTGCTGAACCCCCAGCCGGAACTGACCCCACAAGGCCCTACCGGCGCGGCAGCG(SEQ ID NO:41)

wherein the gene sequence of the VCre recombinase is as follows:

ATGATCGAGAACCAGCTGAGCCTGCTGGGTGATTTCAGCGGCGTGCGTCCGGACGATGTTAAGACCGCGATCCAGGCGGCGCAAAAGAAAGGTATTAACGTTGCGGAGAACGAACAATTCAAAGCGGCGTTTGAGCACCTGCTGAACGAGTTCAAGAAACGTGAGGAACGTTACAGCCCGAACACCCTGCGTCGTCTGGAAAGCGCGTGGACCTGCTTTGTGGATTGGTGCCTGGCGAACCATCGTCACAGCCTGCCGGCGACCCCGGACACCGTTGAGGCGTTCTTTATCGAACGTGCGGAGGAACTGCACCGTAACACCCTGAGCGTGTACCGTTGGGCGATTAGCCGTGTTCATCGTGTTGCGGGTTGCCCGGACCCGTGCCTGGATATCTATGTGGAGGATCGTCTGAAGGCGATTGCGCGTAAGAAAGTGCGTGAGGGCGAAGCGGTTAAACAGGCGAGCCCGTTTAACGAACAACACCTGCTGAAGCTGACCAGCCTGTGGTACCGTAGCGACAAACTGCTGCTGCGTCGTAACCTGGCGCTGCTGGCGGTGGCGTATGAGAGCATGCTGCGTGCGAGCGAACTGGCGAACATCCGTGTTAGCGACATGGAGCTGGCGGGTGATGGCACCGCGATTCTGACCATCCCGATTACCAAGACCAACCACAGCGGCGAGCCGGACACCTGCATTCTGAGCCAGGATGTGGTTAGCCTGCTGATGGACTACACCGAAGCGGGCAAGCTGGACATGAGCAGCGATGGTTTCCTGTTTGTGGGCGTTAGCAAACACAACACCTGCATCAAGCCGAAGAAAGATAAACAGACCGGTGAAGTTCTGCACAAGCCGATTACCACCAAAACCGTGGAGGGCGTTTTCTATAGCGCGTGGGAAACCCTGGATCTGGGTCGTCAAGGCGTGAAGCCGTTTACCGCGCACAGCGCGCGTGTTGGTGCGGCGCAGGACCTGCTGAAGAAAGGCTACAACACCCTGCAAATCCAGCAAAGCGGTCGTTGGAGCAGCGGCGCGATGGTTGCGCGTTATGGTCGTGCGATCCTGGCGCGTGACGGCGCGATGGCGCACAGCCGTGTGAAAACCCGTAGCGCGCCGATGCAATGGGGCAAGGACGAGAAAGATTAA(SEQ ID NO:42)

the amino acid sequence of the VCre recombinase is:

MIENQLSLLGDFSGVRPDDVKTAIQAAQKKGINVAENEQFKAAFEHLLNEFKKREERYSPNTLRRLESAWTCFVDWCLANHRHSLPATPDTVEAFFIERAEELHRNTLSVYRWAISRVHRVAGCPDPCLDIYVEDRLKAIARKKVREGEAVKQASPFNEQHLLKLTSLWYRSDKLLLRRNLALLAVAYESMLRASELANIRVSDMELAGDGTAILTIPITKTNHSGEPDTCILSQDVVSLLMDYTEAGKLDMSSDGFLFVGVSKHNTCIKPKKDKQTGEVLHKPITTKTVEGVFYSAWETLDLGRQGVKPFTAHSARVGAAQDLLKKGYNTLQIQQSGRWSSGAMVARYGRAILARDGAMAHSRVKTRSAPMQWGKDEKD(SEQ ID NO:43)

carrying out PCR amplification by taking the plasmid pK18mobsacB as a template to obtain a vector fragment; amplifying by using a primer and taking the synthesized fragment 10 as a template to obtain a DNA fragment, wherein the fragment contains Bxb1 recombinase gene and an attP sequence corresponding to the recombinase gene, an exogenous gene to be integrated (green fluorescent protein gene GFP in the embodiment) and 2 VloxP sequences specifically recognized by VCre recombinase; according to commercial kits (Gibson)

Master Mix, purchased from New England Biolabs (NEB) Inc.) and the fragments were ligated to the vector fragments by the Gibson Assembly method to obtain the recombinant plasmid pBxb 1-attP-VCre. The primers used are as follows:

the sequence of synthetic fragment 10 was:

CACACAGGAAACAGCTATGACCTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCTACGACATCCCGGTGTGTAGCCGTTCGACCACGCTGCCGAGCCTGAGATGCTGCTCGTACTCTTGCAGATCCCCGAAGTCGATCGTGCGAGTCAGCCCGCCGCGGACGTCGAACGTCAGCCGAACGTTCATCGACCGAAGCCAGGTGTTCTTTGCCGCGGTGTCCTGCTCCCGCCACCAGTCCCCGAACCGCTGCCCGGTCTCGCGCCACTCCCAGCCAGACGGGCGAGCCTCTAGGCCCTCCAGCTCCTCTTGCCGCGCGGCCAGCGCCGCAATACGGGCATCCAGTGCTTCTCGCTGCGGAGAGCCGGCCCGGTAGGCCGGGGAGCCGATCAGCGACGTCAGGTCCACCAGCTCCGCGTTCACCTCCGCGAGTTCGACCGCGGAGTCCGAGCCGGCTACCCAGACTTTCTCCAGACGCTCCGCGTCCCCGAGCAGATCCAGCACCTGCTCCTCGCAGAACGCGTCCCACTCGGCCATCGCCACCGTGCCGTTCCCGCAGTGCTTCGGGAACCCCATCGAGCGGCAGCGGTAGCGCGGGTGCTTACGTCCTCCCCCGGCGAACTTGTACGCGGGCTCCCCGCACACCGCGCAGAACAACACCCGCAGCAGCAGCGACGGGGTAGACACCGCGGGCTTCGCCCGGGAGGTCTTCACGAGCTCGGCGCGCAGCGCCTCCAGCTGCTCACGGGTCAGGATCGGCTCAGCCCGCACCAGCGGGGCTCCGTCGTCGTCTCGGACGGTCTTACCGTTCAGAGTCGCGTACCCGAGCATCGCCTCGGAGATCATCGATCGCTTCAGCGCGGTAGCCGACCACTCCCGGCCCTGCGGCTCGCGGCCTTGCAGCTGCGCGAAGTAGTCCTTCGGCGACAGGACACCACGCCGGTTCAGGTCGTGGGCCACCAGGTGCAGCGGCTCGTGGTTGTCGACGACGCGGTGATACACCTCGAGGATGCGCTCTCGCTGCACAGGGTCCGGCACCAGCCGCCACTCCCCGTCCACGCGCGTAGGCAGGTATCCCCACGGCGGCAGGGATCCTCGGTATTTCCCGGCGCGGATATTGAAATGCGCAGCCGAACGGTTCCGCTCTTTGATCGCTTCTAATTCCATCTGCGCCACCGTTCCCATAAGCGCGATGACGACCGCCGCAAACGGCGTCGTCGTATCGAAGTGCGCTTCGGTCGCGGAGACGACCAGCTTCTTGTGGTCCTCGGCCCAGTGGACCAGCTGTTGCAGATGCCGGATCGATCGGGTCAACCGGTCTACCCGGTACGCCACGATCACGTCGAACGGTTGCTCCTCGAACGCTAGCCACCGGGCCAGGTTCGGTCTGCGCTTCCGGTCGAACGGATCGACCGCCCCGGAGACGTCCAGATCCTCCGCTACCCCGACGACGTCCCAGCCGCGCTGGGCGCAGAGCTGCTGGCAAGACTCCAGCTGACGCTCCGGTGAAGTCGTAGCATCGGTGACGCGGGACAGGCGGATGACTACCAGGGCTCTCATCTAGTATTTCTCCTCTTTCTCTAGTATTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCGCAGGTCAAAGGGTATACTGGGATTCCAGTGAACGCAATCAATTTCTGAGAACTGTCATTCTCGGAAATTGAGGGTTTGTACCGTACACCACTGAGACCGCGGTGGTTGACCAGACAAACCACGAGGGAGACCAGAAACAAAAAAAGGCCCCCCGTTAGGGAGGCCTTCAATAATTGGTTATCATTTGTACAGTTCATCCATACCATGCGTGATGCCCGCTGCGGTTACGAACTCCAGCAGAACCATATGATCGCGTTTCTCGTTCGGATCTTTAGACAGAACGCTTTGCGTGCTCAGATAGTGATTGTCTGGCAGCAGAACAGGACCATCACCGATTGGAGTGTTTTGCTGGTAGTGATCAGCCAGCTGCACGCTGCCATCCTCCACGTTGTGGCGAATTTTAAAATTCGCTTTAATGCCATTTTTTTGTTTATCGGCGGTGATGTAAACATTGTGGCTGTTAAAATTGTATTCCAGCTTATGGCCCAGGATATTGCCGTCTTCTTTAAAGTCAATGCCTTTCAGCTCAATGCGGTTTACCAGGGTATCGCCTTCAAATTTCACTTCCGCACGCGTTTTGTACGTGCCGTCATCCTTAAAGGAAATCGTGCGTTCCTGCACATAGCCTTCCGGCATGGCGGACTTGAAGAAGTCATGCTGCTTCATATGGTCCGGATAACGAGCAAAGCACTGAACACCATAAGTCAGCGTCGTTACCAGAGTCGGCCAAGGTACCGGCAGTTTACCAGTAGTACAGATGAACTTCAGCGTCAGTTTACCATTAGTTGCGTCACCTTCACCCTCGCCACGCACGGAAAACTTATGACCGTTGACATCACCATCCAGTTCCACCAGAATAGGGACGACACCAGTGAACAGCTCTTCGCCTTTACGCATCTAGTATTTCTCCTCTTTCTCTAGTAACTCTTAAACAAAATTATTTGTAGAGGCTGTTTCGTCCTCACGGACTCATCAGACCGGAAAGCACATCCGGTGACAGCTTGCTCGCAGGTCAAAATATATACTGGGATTCCAGTGAACGCAACAGGATGTGACGAGCGGTGTGGTCAATTTCTGAGAACTGTCATTCTCGGAAATTGAACTGGCCGTCGTTTTACAAC(SEQ ID NO:44)

wherein the sequence of VloxP is:

TCAATTTCCGAGAATGACAGTTCTCAGAAATTGA(SEQ ID NO:45)

transferring the recombinant plasmid pBxb1-attP-VCre into Escherichia coli S17-1, transferring the recombinant plasmid into Ralstonia eutropha Bxb1-attB by a conjugative transformation method, and screening positive clones by using an LB plate simultaneously containing 200 mu g/ml kanamycin and 100 mu g/ml apramycin to obtain the recombinant bacteria with plasmids integrated on genomes. Then the recombinant plasmid pVCre is transferred into Escherichia coli S17-1, the recombinant strain is transferred by a joint transformation method, LB plates simultaneously containing 250 mug/ml spectinomycin and 100 mug/ml apramycin are used for randomly picking 8 growing clones, PCR verification is carried out by primers tcggcggcggccgggcgtg (SEQ ID NO:46) and caccgattggagtgttttgc (SEQ ID NO:47), all expected 1547bp bands are obtained, and VCre is proved to delete the vector skeleton (figure 4). And finally, culturing the positive clone on a non-resistance plate to obtain lost pVCre plasmid, namely obtaining recombinant bacteria Ralstonia eutropha Bxb1-GFP with exogenous gene GFP integrated into the genome.

Sequence listing

<110> Shenzhen Lanjing Biotech Ltd

<120> a method for integrating exogenous sequences into genome

<130> DI20-0220-XC37

<160> 47

<170> SIPOSequenceListing 1.0

<210> 1

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H1-primer 1

<400> 1

acacaggaaa cagctatgac tggtacccgg ccaagtctgc 40

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H1-primer 2

<400> 2

gatttgattg tctctctgcc 20

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H2-primer 3

<400> 3

cctgccggcc tggttcaac 19

<210> 4

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H2-primer 4

<400> 4

gttgtaaaac gacggccagt aaagcctcta ccgctcgc 38

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer 5

<400> 5

gtcatagctg tttcctgtgt g 21

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer 6

<400> 6

actggccgtc gttttacaac 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer 7

<400> 7

ggcagagaga caatcaaatc 20

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer 8

<400> 8

gttgaaccag gccggcagg 19

<210> 9

<211> 337

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Synthesis of fragment 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 (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 (Artificial Sequence)

<220>

<223> Synthesis of 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 (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 (Artificial Sequence)

<220>

<223> Synthesis of 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 (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 (Artificial Sequence)

<220>

<223> Synthesis of fragment 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 (Artificial Sequence)

<220>

<223> attB sequence corresponding to P22

<400> 16

acgaccttcg cattacgaat gcgctgc 27

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer 9

<400> 17

cacacaggaa acagctatga c 21

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer 10

<400> 18

gttgtaaaac gacggccagt 20

<210> 19

<211> 1844

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Synthesis of 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 (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 cgcttatggg 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 ccgggagtgg 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 (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 (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 (Artificial Sequence)

<220>

<223> Synthesis of 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 (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 gcagggggcg 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 (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 (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 (Artificial Sequence)

<220>

<223> Synthesis of 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 ttacttaaat 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

ctagctcttt ttgaactttt 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 (Artificial Sequence)

<220>

<223> Gene sequence of TP901 recombinase

<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 agtttactgt 720

ggttatatca aatttaagga cagcctattt gaaggtatgc acaaaccaat tatcccttat 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 (Artificial Sequence)

<220>

<223> TP901 recombinase

<400> 29

Met Lys His 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 (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 (Artificial Sequence)

<220>

<223> Synthesis of 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 tatttttact gtgggacata tttgggacag 1680

aagtaccaaa aaactggccg tcgttttaca ac 1768

<210> 32

<211> 1164

<212> DNA

<213> Artificial Sequence (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 (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 (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 (Artificial Sequence)

<220>

<223> primer

<400> 35

gcgcatggcg tctccatg 18

<210> 36

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 36

gtggaccagc tgttgcag 18

<210> 37

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer 11

<400> 37

ctaccggcgc ggcagcg 17

<210> 38

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer 12

<400> 38

gcggccaccg gctggctc 18

<210> 39

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer 13

<400> 39

cgctgccgcg ccggtag 17

<210> 40

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Primer 14

<400> 40

gagccagccg gtggccgc 18

<210> 41

<211> 5555

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Synthesis of 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

atgactgttt ttttggggta 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 (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 (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 (Artificial Sequence)

<220>

<223> Synthesis of 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 (Artificial Sequence)

<220>

<223> sequence of VloxP

<400> 45

tcaatttccg agaatgacag ttctcagaaa ttga 34

<210> 46

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 46

tcggcggcgg ccgggcgtg 19

<210> 47

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 47

caccgattgg agtgttttgc 20

Claims (10)

1. A gene insertion method based on site-specific recombinase suitable for eubacterium rolfsii comprises the following steps:

1) inserting an attB site corresponding to a site-specific recombinase Bxb1 into the genome of the Eubacterium rolfsii to construct a recombinant Eubacterium rolfsii with an attB sequence integrated on 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 Bxb 1;

3) transferring the recombinant vector constructed in the step 2) into the recombinant Rogowski eubacterium constructed in the step 1), and mediating recombination between attB and attP sequences by using the site-specific recombinase Bxb1, thereby integrating the recombinant vector into the genome of the recombinant Rogowski eubacterium.

2. The method for inserting a gene based on a site-specific recombinase suitable for Eubacterium rolfsii according to claim 1, comprising the steps of:

a) inserting an attB site corresponding to a site-specific recombinase Bxb1 into the genome of the Eubacterium rolfsii to construct a recombinant Eubacterium rolfsii with an attB sequence integrated on the genome;

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

c) constructing a recombinant vector containing an exogenous sequence, a site-specific recombinase Bxb1 gene, attP sequence corresponding to the site-specific recombinase Bxb1, and 2 VloxP sequences capable of being specifically recognized by VCre recombinase in step b), wherein the exogenous sequence and the attP sequence are between the 2 VloxP sequences, and the site-specific recombinase Bxb1 gene is not between the 2 VloxP sequences;

d) transferring the recombinant vector constructed in the step c) into the recombinant Rogowski bacterium constructed in the step a), and mediating recombination between attB and attP sequences by using the site-specific recombinase Bxb1, thereby integrating the recombinant vector into the genome of the recombinant Rogowski bacterium;

e) transferring the recombinant vector constructed in the step b) into the recombinant bacterium integrated with the recombinant vector on the genome obtained in the step d), so as to delete the skeleton part of the recombinant vector from the genome.

3. The method of claim 1 or 2, wherein the eubacterium rolfsii is Ralstonia eutropha H16.

4. The method according to claim 1 or 2, wherein in step 2) or c) the recombinant vector is a plasmid vector incapable of replication in eubacterium rolfsii; preferably, the backbone portion of the recombinant vector comprises a replicon capable of replicating in E.coli (e.g., S17-1) but incapable of replicating in Eubacterium rolfsii, preferably the replicon is selected from the group consisting of a pMB1 replicon, a pUC replicon, a p15a replicon and a R6K γ replicon, more preferably the replicon is a pMB1 replicon; preferably, the backbone portion of the recombinant vector further comprises a selectable marker gene; more preferably, the selection marker gene is an antibiotic resistance gene, particularly, the antibiotic resistance gene is selected from the group consisting of a kanamycin resistance gene, a tetracycline resistance gene, a streptomycin resistance gene, and a spectinomycin resistance gene, and more particularly, the antibiotic resistance gene is a kanamycin resistance gene.

5. The method according to claim 2, wherein in step b), preferably, the recombinant vector may be a plasmid vector capable of replicating in Eubacterium rolfsii; preferably, the backbone portion of the recombinant vector comprises a replicon capable of replicating both in E.coli and in Eubacterium rolfsii, preferably the replicon is selected from the group consisting of a pBBR1 replicon, an SC101 replicon, and a RK2 replicon, more preferably the replicon is a pBBR1 replicon; preferably, the backbone portion of the recombinant vector further comprises a selectable marker gene; more preferably, the selection marker gene is an antibiotic resistance gene, preferably, the antibiotic resistance gene is selected from the group consisting of kanamycin resistance gene, tetracycline resistance gene, streptomycin resistance gene and spectinomycin resistance gene, and more preferably, the antibiotic resistance gene is kanamycin and spectinomycin resistance gene.

6. The method of claim 1 or 2,

in the step 1) or a), the sequence of attB site corresponding to the site-specific recombinase Bxb1 is shown as SEQ ID NO. 10; and/or

In the step 2) or c), the amino acid sequence of the site-specific recombinase Bxb1 gene is shown as SEQ ID NO. 20; and/or

In the step 2) or c), the attP sequence corresponding to the site-specific recombinase Bxb1 is shown as SEQ ID NO. 22; and/or

In the step b), the amino acid sequence of the VCre recombinase is shown as SEQ ID NO 42; and/or

In step c), preferably, the VloxP sequence is shown in SEQ ID NO: 44.

7. The method according to claim 1 or 2, wherein in step 2) or c), the nucleotide sequence of the site-specific recombinase Bxb1 gene is shown as SEQ ID NO 21; and/or, in the step b), the nucleotide sequence of the VCre recombinase is shown as SEQ ID NO 41.

8. A recombinant vector comprising an exogenous sequence, a site specific recombinase Bxb1 gene, an attP sequence corresponding to site specific recombinase Bxb 1.

9. The recombinant vector according to claim 8, wherein said recombinant vector further comprises 2 VloxP sequences capable of being specifically recognized by VCre recombinase, wherein said exogenous sequence and said attP sequence are between said 2 VloxP sequences, and said site-specific recombinase Bxb1 gene is not between said 2 VloxP sequences.

10. A recombinant vector comprising a VCre recombinase gene.

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