CN106929528B - Novel recombination system in pseudomonas and application thereof - Google Patents

Abstract

The invention discloses a novel recombination system in Pseudomonas, which is based on an operon (Orf38, Orf37 and Orf36) from Pseudomonas aeruginosa (Pseudomonas aeruginosa) phage Ab31 similar to lambda Red, and an operon (rect) from Pseudomonas syringae B728a prophage pv. syringae B728a similar to RecET_Psy) And (5) constructing. The recombination system comprises two genes of host specificity exonuclease and single-stranded DNA annealing protein, wherein the third gene is exonuclease recBCD inhibitor protein, and the third gene or the fourth gene can also be single-stranded DNA binding protein SSB from bacterial chromosome, bacterial plasmid and other sources. The function detection result shows that the hybrid system consisting of the lambda Red-like operon, the RecET-like operon and the Red gamma/Plu gamma can work in pseudomonas, and the SSB protein can remarkably improve the recombination efficiency. The recombination systems can be used for carrying out efficient and concise plasmid modification, point mutation and insertion deletion genome modification on pseudomonas, and have wide application prospect.

Description

Novel recombination system in pseudomonas and application thereof

Technical Field

The invention relates to a recombination system in gram-negative bacillus and construction and application thereof, in particular to a recombination system in pseudomonas aeruginosa, pseudomonas syringae, pseudomonas fluorescens or/and pseudomonas putida and construction and application thereof, belonging to the field of microbial genetic engineering.

Background

The recombination system of phage coding can be widely applied in modifying prokaryotic genome and constructing eukaryotic transgenic species. It only needs 35 nucleotides as homology arms, and the application mainly comprises the construction of mutants-point mutation, deletion, insertion and substitution. The shorter homology arm is the main advantage of this technique, which is named recombineering.

In E.coli, the most widely used recombination systems are the lambda Red system and the racRecET system. The lambda Red system uses Red operons (Red α, Red β and Red γ) from lambda phage to mediate homologous recombination of linear and circular DNA molecules. The racRecET system uses the RecE and RecT proteins of Rac prophages to mediate homologous recombination of two linear DNA molecules in E.coli. Red alpha and RecE are 5 '-3' double-stranded DNA exonucleases, and Red beta and RecT are single-stranded annealed proteins. The literature reports that Red α/Red β and RecE/RecT promote homologous recombination of double-stranded DNA through protein-protein interactions.

Red γ inhibits exonuclease activity of RecBCD, a multifunctional enzyme complex that processes DNA ends generated by double strand breaks. Red γ can form a dimeric DNA analog that interacts with and inhibits the activity of exonucleases and helicases in the RecBCD system. The addition of Red gamma to the RecE/RecT or Red alpha/Red beta system has been reported in the literature to improve DNA recombination efficiency. Plu gamma from Photorhabdus luminescens (Photorhabdus luminescens) can also imitate the shape and structure of double-helix DNA, and inhibit the exonuclease complex Plu0632/Plu0630/Plu0633 in a host body, so that the efficiency of homologous recombination can be effectively improved in vivo.

Similar to the construction of the lambda Red system and the racRecET system, an operon containing exonuclease and single-stranded annealing protein is searched and cloned in phage or prophage genomes of other bacteria, a Red gamma or Plu gamma protein gene capable of improving recombination efficiency is inserted into the operon to construct a complete DNA recombination system, and the recombination system is introduced into a host from the operon through electric shock transformation or conjugal transfer for genetic modification after the construction in Escherichia coli is completed. Many documents report recombination systems constructed by using recombinant functional operons of bacteriophage, such as photobacterium, Xenorhabdus (Xenorhabdus), bacillus subtilis (b.subtilis), Clostridium (Clostridium), pseudomonas syringae (p.syringae), mycobacterium tuberculosis (m.tuberculosis), Lactobacillus (Lactobacillus plantarum), and the like, but host specificity results in a narrow range of applications of these recombination systems.

The most studied pseudomonads are the human conditional pathogens Pseudomonas aeruginosa (Pseudomonas aeruginosa), the plant conditional pathogens Pseudomonas syringae (Pseudomonas syringae), the plant growth promoting bacteria Pseudomonas fluorescens (Pseudomonas fluorescens), and also the Pseudomonas putida (Pseudomonas putida) frequently used in bioremediation and biocontrol processes. The traditional gene operation technology is to realize the modification of insertion, knockout, point mutation and the like of a target gene in vitro by using technologies such as restriction enzyme, DNA ligase, PCR amplification and the like, but has the defects of complicated operation and low efficiency, and research and modification of a pseudomonas genome are limited due to the defects of restriction enzyme sites, complicated operation, low efficiency and the like. Researches and patents about recombinant systems in pseudomonas aeruginosa, pseudomonas syringae, pseudomonas fluorescens and pseudomonas putida and construction and application of the recombinant systems are rarely reported.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a recombination system in pseudomonas (pseudomonas aeruginosa, pseudomonas syringae, pseudomonas fluorescens or/and pseudomonas putida) and construction and application thereof.

In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:

The novel recombination system in the pseudomonas is composed of a series of homologous recombination system expression plasmids, and is characterized in that: the expression plasmid expresses an operon under an inducible promoter, and the operon comprises two genes of host-specific exonuclease and single-stranded DNA annealing protein, wherein the third gene is Red gamma derived from Escherichia coli lambda phage, or Plu gamma derived from Prophage of photobacterium (Photorhabdus luminescens) (Plu2934), or other exogenous exonuclease recBCD inhibitor protein; alternatively, the operon comprises three genes, a host-specific exonuclease, a single-stranded DNA annealing protein and an exogenous or endogenous exonuclease recBCD suppressor protein, and the fourth gene is a single-stranded DNA binding protein SSB derived from the bacterial chromosome, or an SSB derived from a bacterial plasmid, or an SSB derived from a bacteriophage, or an SSB derived from a bacterial prophage.

In the novel recombination system in the pseudomonas, the technical scheme for further optimization is as follows: the expression plasmid of the serial homologous recombination system in the recombination system comprises a pBBR1 replication origin, a kanamycin resistance gene and a recombination system of a rhamnose inducible promoter; the names are respectively pBBR1-Rha-red _ gba-Kan, and the nucleotide sequence is shown in SEQ ID No. 23; pBBR1-Rha-plu _ gba-kan, the nucleotide sequence is shown as SEQID No. 24; pBBR1-Rha-TEpsy-kan, the nucleotide sequence is shown in SEQ ID No. 25; pBBR1-Rha-pluGam-TEpsy-kan, the nucleotide sequence is shown in SEQ ID No. 26; pBBR1-Rha-redGam-TEpsy-kan, the nucleotide sequence is shown in SEQ ID No. 27; pBBR1-Rha-BAS-kan, the nucleotide sequence is shown in SEQ ID No. 28; pBBR1-Rha-pluGam-BAS-kan, the nucleotide sequence is shown in SEQ ID No. 29; pBBR1-Rha-redGam-BAS-kan, the nucleotide sequence is shown in SEQ ID No. 30; wherein, RecTEpsy is RecTEpsy in prophage from Pseudomonas syringae (Pseudomonas syringae), B is single-stranded DNA annealing protein or recombinase, A is 5 '-3' double-stranded DNA exonuclease, S is single-stranded DNA binding protein (SSB) from Pseudomonas aeruginosa bacteriophage vB _ PaeP _ Tr60_ Ab31, Red gamma is suppressor protein of Escherichia coli exonuclease recBCD from Lambda phage, Plu gamma is suppressor protein of exonuclease recBCD from prophage of Photorhabdus luminescens (Photorhabdus luminescens).

In the novel recombination system in the pseudomonas, the optimized technical scheme is as follows: the expression plasmid of the series of homologous recombination systems in the recombination system is as follows: pBBR1-Rha-BAS-kan, pBBR1-Rha-pluGam-TEpsy-kan, pBBR1-Rha-redGam-TEpsy-kan, pBBR1-Rha-redGam-BAS-kan or pBBR 1-Rha-pluGam-BAS-kan.

In the novel recombination system in pseudomonas, the pseudomonas is preferably pseudomonas aeruginosa, pseudomonas syringae, pseudomonas fluorescens or/and pseudomonas putida.

The novel recombination system in the pseudomonas is applied to the genetic modification of genome DNA or plasmid by mediating the homologous recombination between a short homologous arm and a long homologous arm in different species of pseudomonas. Wherein, the short homology arm refers to a homology arm of 50bp-150bp, and the long homology arm refers to a homology arm of more than 150 bp.

The optimal recombination systems in various strains are different, the optimal recombination conditions are different, condition optimization such as optimization of a competent cell preparation method, optimization of DNA conversion amount, optimization of antibiotic selection concentration, optimization of homology arm length and the like can be carried out to improve the recombination efficiency, and efficient gene modification is further carried out by utilizing the optimal recombination systems of different strains.

In the genetic modification of the genomic DNA or plasmid by the above-mentioned mediation of homologous recombination between the short and long homology arms, the plasmid pBBR1-Rha-redGam-BAS-kan is preferable in Pseudomonas aeruginosa (Pseudomonas aeruginosa), the plasmid pBBR1-Rha-redGam-BAS-kan is preferable in Pseudomonas syringae (Pseudomonas aeruginosa), the plasmid pBBR1-Rha-redGam-BAS-kan is preferable, the plasmid pBBR 1-Rha-redGam-TESy-kan or the plasmid pBBR 1-Rha-pluGam-TESy-kan is preferable in Pseudomonas putida (Pseudomonas putida), the plasmid pBBR1-Rha-BAS-kan is preferable, and the plasmid pBBR1-Rha-BAS or the plasmid Rbr 1-Rhamn is preferable in Pseudomonas fluorescens (Pseudomonas fluorescens).

The invention reports a series of recombination systems which can be applied to pseudomonas, in particular to the construction of the optimal recombination system in pseudomonas aeruginosa, pseudomonas syringae, pseudomonas fluorescens and pseudomonas putida, wherein the recombination systems are two host-specific operons with recombination functions from pseudomonas aeruginosa phage Ab31 and pseudomonas syringae prophage. BLAST analysis showed that Orf38, Orf37, and Orf36 are homologous proteins to Red β, Red α, and single-stranded DNA binding protein (SSB), respectively. The recombination experiment result shows that the hybrid system with Red gamma/Plu gamma can work well in some pseudomonads, and the SSB protein is found to improve the recombination efficiency obviously. By utilizing the recombination systems, efficient and concise plasmid modification, point mutation and insertion deletion genome modification are successfully carried out on pseudomonas, and the application prospect is wide.

Drawings

FIG. 1: comparison of recombination efficiencies of different recombinase combinations in Pseudomonas.

(A) Schematic representation of the recombination of the loop in Pseudomonas. The PCR product is a new plasmid formed by replacing recombinant related genes in an expression plasmid with a gentamycin resistance gene (Genta) in the middle and with homologous arms (blue) with 100bp at two ends.

(B) Pseudomonas aeruginosa expressed pBBR1-Rha-red _ gba-Kan, pBBR1-Rha-plu _ gba-Kan, pBBR1-Rha-TEpsy-Kan, pBBR1-Rha-pluGam-TEpsy-Kan, pBBR1-Rha-redGam-TEpsy-Kan, pBBR1-Rha-BAS-Kan, pBBR1-Rha-pluGam-BAS-Kan, pBBR1-Rha-RedGam-BAS-Kan at 30 ℃ for the results of the comparison of the efficiency of the wire loop recombination.

(C) And (3) expressing a series of recombination systems in the step (B) by using the pseudomonas aeruginosa under the condition of 37 ℃ to compare the efficiency of the wire loop recombination.

(D) The pseudomonas fluorescens expresses a series of recombination systems in the step (B) at the temperature of 30 ℃ to compare the efficiency of the wire loop recombination.

(E) The pseudomonas syringae expresses a series of recombination systems in the step (B) at the temperature of 30 ℃ to compare the efficiency of the wire loop recombination.

(F) Pseudomonas putida expresses pBBR1-Rha-red _ gba-Kan, pBBR1-Rha-plu _ gba-Kan, pBBR1-Rha-TEpsy-Kan, pBBR1-Rha-pluGam-TEpsy-Kan, pBBR1-Rha-redGam-TEpsy-Kan, pBBR1-Rha-BAS-Kan, pBBR1-Rha-pluGam-BAS-Kan, pBBR1-Rha-RedGam-BAS-Kan at 30 ℃ for the results of comparison of the efficiency of the wire loop recombination.

FIG. 2: and (5) optimizing the pseudomonad electrotransfer condition.

(A) and (B), (C) and (D) are growth curves for Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas syringae and Pseudomonas fluorescens. The initial bacterial suspension concentration OD600 was 0.085.

(E) (F), (G) and (H) are the transformation efficiencies of Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas syringae and Pseudomonas fluorescens respectively at different times.

FIG. 3: optimization of the amount of transforming DNA and the length of the homology arm upon recombination in Pseudomonas.

the influence of transforming different concentrations of DNA on the recombination efficiency in Pseudomonas aeruginosa carrying pBBR1-Rha-pluGam-TEpsy-kan (A), Pseudomonas putida carrying pBBR1-Rha-BAS-kan system (B), Pseudomonas syringae carrying pBBR1-Rha-BAS-kan (C), and Pseudomonas fluorescens carrying pBBR1-Rha-redGam-TEpsy-kan (D).

Effect of transforming PCR products of different length homology arms in pBBR1-Rha-redGam-TEpsy-kan (H) on recombination efficiency in Pseudomonas aeruginosa carrying pBBR1-Rha-pluGam-TEpsy-kan (E), Pseudomonas putida carrying pBBR1-Rha-BAS-kan (F), Pseudomonas syringae carrying pBBR1-Rha-BAS-kan (G), and Pseudomonas fluorescens carrying pBBR1-Rha-redGam-TEpsy-kan (H).

FIG. 4: PCR identification of colonies after genome modification by homologous recombination in P.aeruginosa.

(A) Schematic representation of genome modification in pseudomonas aeruginosa and schematic representation of colony PCR.

(B) By inserting a gentamicin selection marker to knock out the phnAB gene, clones 1 and 2 were both correct.

(C) Six clones were correct after knocking out the phnAB gene by deleting the gentamicin resistance selection marker using site-specific recombination by Cre enzyme.

(D) Both clones 1 and 2 were correct by knocking out the lasB gene by inserting the gentamicin selection marker.

(E) After knocking out the lasB gene, the gentamicin resistance selection marker was deleted using site-specific recombination with Cre enzyme and the cloning was correct.

(F) The mdoH gene was knocked out by inserting a gentamicin selection marker and the cloning was correct.

(G) After the mdoH gene was knocked out, the gentamicin resistance selection marker was deleted using site-specific recombination of Cre enzyme, and all four clones were correct.

FIG. 5: point mutations were performed in pseudomonas aeruginosa using a novel recombination system.

(A) Schematic representation of point mutations and colony PCR identification in pseudomonas aeruginosa.

(B) The SacB-gentamicin module was inserted at the toxA target site, lane C was the wild type strain as a negative control, lane M was TakaraDL5000, lanes 1 and 2 were recombinants, and both clones 1 and 2 were correct.

(C) Three clones were correct by single-stranded nucleotide paired insertion point mutation.

(D) And (5) sequencing and verifying the mutant.

FIG. 6: functional validation of SacB was performed on LB (left panel) and LB (right panel) containing 10% sucrose.

1. A wild-type strain of pseudomonas aeruginosa; different mutant clones after insertion of PAO1 into the SacB-Gentamicin module.

FIG. 7: PCR verification of genetically modified colonies was performed in Pseudomonas syringae and Pseudomonas fluorescens. The PCR product spans the entire insert.

(A) insertion of promoter (P) before the predicted biosynthetic genes of Pseudomonas syringae_genta)。

(B) the retS gene of P.fluorescens Pf5 was knocked out, lane 1 was the final mutant strain (Pf5- Δ retS) after site-specific recombination with Cre enzyme, with the gentamycin-SacB module (Pf5- Δ retS-genta-loxM) inserted therein.

FIG. 8: effect of SSB on the recombination function of the pBBR1-Rha-BA-kan and pBBR1-Rha-redGam-BA-kan systems.

The effect of SSB on the recombination function of pBBR1-Rha-BA-kan and pBBR1-Rha-redGam-BA-kan was investigated in E.coli (A), P.aeruginosa (B) and P.syringae (C).

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples, wherein the methods are not specifically described, and conventional methods are employed, and reagents such as conventional commercially available reagents or reagents prepared by conventional methods are employed.

Reagents and instrumentation:

In the present example, the reagents were mainly molecular biology experimental reagents, single-stranded nucleotides were synthesized from Shanghai Biotechnology, Inc., restriction enzymes, DNA polymerases, and DNA tags were obtained from New England Biolabs, and antibiotics were purchased from Invitrogen.

Preparation of the cells for recombinant electrotransformation:

Coli GB05, Pseudomonas aeruginosa PAO1, DSMZ, Germany (accession No.: DSM 1707), and Pseudomonas fluorescens pf5 were transformed with the respective plasmids and were obtained from American type culture Collection ATCC (accession No.:BAA-477^TM) Pseudomonas syringae DC3000 was purchased from american type culture collection ATCC (accession number:BAA-871^TM) Pseudomonas putida KT2440 was purchased from DSMZ (accession number: DSM 6125). Coli competent cells were prepared according to the procedures reported in the literature. Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas syringae, Pseudomonas putida, strains with expression plasmids were first cultured overnight and then diluted to 1.3ml LB medium (Pseudomonas syringae in KB medium), the initial OD₆₀₀Is about 0.085. Culturing at 950rpm to start logarithmic phase, adding inducer L-rhamnose to final concentration of 2.5g L^-1The cells were incubated at 30 ℃ or 37 ℃ for 40 minutes at 950 rpm. When preparing competent cells, the strain solution 9500rpm was centrifuged at room temperature for 30 seconds, the supernatant was discarded, and 1ml of ddH was added₂Resuspend and centrifuge, repeat this step once. Next, the cells were resuspended in 30. mu.l ddH₂And adding DNA into the mixture, adding 1500ng of PCR product when performing plasmid modification, and adding 2.5 mu g of PCR product when performing genome modification on pseudomonas aeruginosa, pseudomonas fluorescens, pseudomonas syringae and pseudomonas putida. The electric rotor used a 1mm gauge, electric converter (Eppendorf 2510 Electropotato)r) the voltage was set to 1250V. After the electrotransformation is completed, the cells are recovered for 100 minutes at 30 ℃ or 60 minutes at 37 ℃, and finally coated on plates with proper antibiotic concentration, the pseudomonas aeruginosa, the pseudomonas fluorescens and the pseudomonas putida can be counted on the second day, and the pseudomonas syringae can be counted on the third or fourth day.

Example 1: construction of a series of homologous recombination expression plasmids

BLAST analyses of genomes from Pseudomonas and Pseudomonas phages with the amino acid sequence of lambda Beta or rac RecT as a reference, look for potential proteins with recombinant function. Two operons containing paired exonuclease-recombinases were found from pseudomonas and pseudomonas phages, one operon being RecTEpsy from pseudomonas syringae, which has been successfully used to construct recombinant systems; the second operon derived from the Pseudomonas phage vB _ PaeP _ Tr60_ Ab31, this operon encodes three proteins, Orf38(B) contains 177 amino acids, and the sequence has 62% homology with Red β; orf37(a) contains 195 amino acids, the sequence has 32% homology to Red α; orf36(S) contains 148 amino acids and has a sequence with 54% homology to the single-stranded DNA binding protein SSB of E.coli.

A series of recombinant functional plasmids are constructed on the basis of a wide-host replicon pBBR1, and recombinant functional proteins are induced and expressed by a rhamnose promoter.

Construction method of pBBR1-Rha-red _ gba-kan plasmid: extracting pBBR1-Rha-GFP-kan plasmid from escherichia coli, cutting 40ul of plasmid by NdeI for 3h, carrying out gel running by agarose gel electrophoresis of 90V until the fragments run to the bottom of gel, then carrying out gel recovery of linear vector, amplifying red _ gba fragment by taking pSC101-BAD-gba-tet plasmid and gba-3/gba-5 as a template, carrying out overnight culture and transfer of GB-dir bacteria at 30 ℃ for 2h, adding 20ul of arabinose inducer for 40min at 37 ℃, carrying out electric shock co-transformation of pBBR1-Rha-kan linear vector and red _ gba fragment 1350V into the induced GB05-dir, recovering the bacteria at 37 ℃ for 1h, centrifuging the bacteria at 10000rpm, streaking the bacteria onto a resistant plate with kanamycin 15, carrying out overnight culture and single clone identification at 37 ℃, and correctly cloning for bacterial conservation.

Other plasmids were constructed analogously: the pBBR1-Rha-plu _ gba-kan plasmid is obtained by cotransformation of a pBBR1-Rha-kan linear vector and a plu _ gba fragment into GB05-dir through electric shock and linear recombination through arabinose induction; the pBBR1-Rha-TEpsy-kan plasmid consists of pBBR1-Rha-kan linear vector and TE_PsyThe fragment is transformed into GB05-dir by electric shock, and the arabinose is induced to carry out linear recombination to obtain the product; the pBBR1-Rha-pluGam-TEpsy-kan plasmid is obtained by cotransformation of a pBBR1-Rha-TEpsy-kan linear vector and a pluGam fragment into GB05-dir through electric shock and linear recombination through arabinose induction; the pBBR1-Rha-redGam-TEpsy-kan plasmid is obtained by cotransformation of a pBBR1-Rha-TEpsy-kan linear vector and a redGam fragment into GB05-dir through electric shock and linear recombination through arabinose induction; the pBBR1-Rha-BAS-kan plasmid is obtained by cotransformation of a pBBR1-Rha-kan linear vector and a BAS fragment into GB05-dir through electric shock, and linear recombination is carried out through arabinose induction; the pBBR1-Rha-pluGam-BAS-kan plasmid is obtained by cotransformation of a pBBR1-Rha-BAS-kan linear vector and a pluGam fragment into GB05-dir through electric shock and linear recombination through arabinose induction; the pBBR1-Rha-redGam-BAS-kan plasmid is obtained by cotransformation of pBBR1-Rha-BAS-kan linear vector and redGam fragment into GB05-dir through electric shock and linear recombination through arabinose induction.

in addition to the two genes of host-specific exonuclease and single-stranded DNA annealing protein, or in addition to the three genes of host-specific exonuclease, single-stranded DNA annealing protein and exogenous or endogenous exonuclease recBCD suppressor protein, the third gene or the fourth gene may also be a single-stranded DNA binding protein SSB derived from a bacterial chromosome, or an SSB derived from a bacterial plasmid, or an SSB derived from a bacterial bacteriophage, or an SSB derived from a bacterial prophage.

Example 2: comparison of the efficiency of different recombination systems in Pseudomonas

pBBR1-Rha-red _ gba-Kan, pBBR1-Rha-plu _ gba-Kan, pBBR1-Rha-TEpsy-Kan, pBBR1-Rha-pluGam-TEpsy-Kan, pBBR1-Rha-redGam-TEpsy-Kan, pBBR1-Rha-BAS-Kan, pBBR1-Rha-pluGam-BAS-Kan, pBBR1-Rha-RedGam-BAS-Kan, and pBBR1-Rha-RedGam-BAS-Kan were introduced into four kinds of Pseudomonas (Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas fluorescens, and Pseudomonas putida) by electric shock transformation. FIG. 1A is a schematic representation of the efficiency comparison of a recombination system. P. aeruginosa has good growth conditions at both 37 ℃ and 30 ℃ and therefore an efficiency comparison between these two temperatures is required. P. aeruginosa itself has multiple resistance and it is easy to obtain a stronger resistance mechanism, so the accuracy is calculated after the recombination efficiency comparison in P.aeruginosa.

In P.aeruginosa, the addition of Red gamma and Plu gamma proteins to the BAS recombination system significantly improved the accuracy of homologous recombination, but for the pBBR1-Rha-TEpsy-kan plasmid, the accuracy was improved only by the addition of Plu gamma protein at 30 ℃ in culture (FIG. 1B). The pBBR1-Rha-BAS-kan plasmid works when cultured at 37 ℃, and the accuracy of gene recombination can be remarkably improved after the addition of Red gamma protein (FIG. 1C). We therefore used the pBBR1-Rha-pluGam-TEpsy-kan plasmid at 30 ℃ and the pBBR1-Rha-RedGam-BAS-kan plasmid at 37 ℃ for genetic modification in P.aeruginosa. Similar to that of P.aeruginosa, the addition of Red. gamma. and Plu. gamma. proteins to the plasmids pBBR1-Rha-TEpsy-kan and pBBR1-Rha-BAS-kan in P.fluorescens also significantly increased the efficiency of recombination (FIG. 1D). However, in P.syringae and P.putida, addition of Red. gamma. and Plu. gamma. proteins to the pBBR1-Rha-BAS-kan plasmid reduced the recombination efficiency (FIG. 1E, 1F).

Finally, it was determined that the recombinant expression plasmids of high efficiency in Pseudomonas (Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas fluorescens and Pseudomonas putida) were pBBR1-Rha-BAS-kan plasmid, pBBR1-Rha-redGam-TEpsy-kan plasmid, pBBR1-Rha-pluGam-TEpsy-kan plasmid, pBBR1-Rha-pluGam-BAS-kan plasmid and pBBR1-Rha-RedGam-BAS-kan plasmid. Wherein S is a single stranded DNA binding protein (SSB) derived from bacteriophage vB _ PaeP _ Tr60_ Ab31, Red γ is an inhibitor protein of the e.coli exonuclease recBCD derived from lambda phage, Plu γ is an inhibitor protein of the exonuclease recBCD derived from Photorhabdus luminescens prophage.

Example 3: optimization of the working conditions of the recombination system in pseudomonas so as to improve the recombination efficiency

According to the embodiment 2, the optimal recombination system is different for each pseudomonas, and the recombination systems can improve the recombination efficiency on the basis of optimizing the preparation method of competent cells, optimizing the DNA transformation amount, optimizing the antibiotic selection concentration and optimizing the length of a homology arm in different pseudomonas.

the ability of exogenous DNA to enter cells by electric shock has a great relationship with the efficiency of homologous recombination of DNA molecules, and the optimal period of exogenous DNA to enter cells by electric shock is an exponential growth phase. The growth curves of Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas syringae and Pseudomonas fluorescens were determined in the present invention (FIG. 2 ABCD). The data show that exponential growth phases of pseudomonas aeruginosa, pseudomonas putida, pseudomonas syringae and pseudomonas fluorescens begin at 2h, 2h, 3h and 1h, respectively. From the measured growth curve, the optimal time for transformation can be analyzed. The results show that the optimal transformation times for P.aeruginosa, P.putida, P.syringae and P.fluorescens were 2.5h, 2.5h, 4h and 1.5h, respectively (FIG. 2 EFGH).

The invention simultaneously detects whether the concentration of the transforming DNA can influence the gene recombination efficiency. Pseudomonas aeruginosa carried pBBR1-Rha-pluGam-TEpsy-kan plasmid, Pseudomonas putida carried pBBR1-Rha-BAS-kan plasmid, Pseudomonas syringae carried pBBR1-Rha-BAS-kan plasmid, Pseudomonas fluorescens carried pBBR1-Rha-redGam-TEpsy-kan plasmid, were transformed to quantify the same volume of PCR product (gentamycin resistance gene) respectively, where the amount of DNA was a gradient of 100ng, 250ng, 500ng, 1000ng, 1500ng and 2000ng, and then the recombination efficiency was counted. Detection of DNA concentration by this gradient showed that the amount of saturated DNA in Pseudomonas for recombination was 1500ng (FIG. 3 ABCD).

The invention also studies the influence of the length of the homology arm on the efficiency of homologous recombination. Pseudomonas aeruginosa carries pBBR1-Rha-pluGam-TEpsy-kan plasmid, Pseudomonas putida carries pBBR1-Rha-BAS-kan plasmid, Pseudomonas syringae carries pBBR1-Rha-BAS-kan plasmid, Pseudomonas fluorescens carries pBBR1-Rha-redGam-TEpsy-kan plasmid, and PCR products (gentamycin resistance genes) with different lengths of homology arms are respectively transferred by electric shock. Can be classified into two types in the pseudomonas aeruginosa, one type is that the length of a homologous arm is less than 50bp, and basically has no recombination function (figure 3E); the second type is a homology arm length from 75bp to 150bp, in this length range, recombination efficiency is significantly improved (FIG. 3E). The shortest homology arm lengths for P.putida, P.syringae and P.fluorescens were 75bp (FIG. 3F), 50bp (FIG. 3G) and 100bp (FIG. 3H), respectively.

Example 4: genetic modification of genomes in different pseudomonads using respective optimized novel recombination systems

The invention successfully establishes an optimal recombination system for different pseudomonas, optimizes the working conditions of the series of recombination systems, and then carries out genetic modification on the genomes of different pseudomonas by utilizing the optimal recombination systems. The homologous recombination between a short homology arm (50bp-150bp) and a long homology arm (more than 150bp) is mediated in different species of pseudomonas to carry out genetic modification of genome DNA. The plasmid pBBR1-Rha-pluGam-BAS-kan is preferred in Pseudomonas aeruginosa, the plasmid pBBR1-Rha-RedGam-BAS-kan, the plasmid pBBR1-Rha-redGam-TEpsy-kan or the plasmid pBBR1-Rha-pluGam-TEpsy-kan is preferred in Pseudomonas putida, the plasmid pBBR1-Rha-BAS-kan is preferred in Pseudomonas fluorescens, and the plasmid pBBR1-Rha-BAS-kan or the plasmid pBBR1-Rha-pluGam-TEpsy-kan is preferred in Pseudomonas fluorescens.

Genome modification of pseudomonas aeruginosa PAO1 includes gene knockout and gene knock-in. FIG. 4A shows a schematic diagram of the gene knockout process, in which a PCR product is first inserted into a target gene by homologous recombination, the PCR product includes a gentamicin resistance gene, a homology arm (75bp) and a site-specific recombinase recognition site loxM (lox71-lox66), and the target gene is: (ii) a The phnAB gene, which is an essential gene for biosynthesis of pyocyanin; the lasB gene, which encodes elastase B; the mdoH gene, which encodes a protein involved in periplasmic glucan biosynthesis. After the gene recombination, the colony PCR at the 5 '-3' end was used for verification (FIG. 4 BCD). Approximately 100 correct gentamicin resistant clones were obtained by a single electrotransformation with the plasmid pBBR 1-Rha-pluGam-TEpsy-kan. However, when the plasmid pBBR1-Rha-RedGam-BAS-kan was used for genetic modification, the accuracy was lowered. To obtain a traceless modified mutant of P.aeruginosa PAO1, the resistance gene was knocked out by site-specific recombination by Cre enzyme, and finally colony PCR identification was performed on each mutant (FIG. 4 EFG).

ToxA has three domains: the amino-terminal domain I comprises a recognition site for eukaryotic cells, the intermediate domain II comprises protein transfer functions, and the carboxy-terminal domain III comprises a binding site for biological enzymes. Glutamic acid 578 in domain III is the active site and substitution of this amino acid can create an attenuated strain. The point mutation of ToxA (changing codon 578 from GAG to GAC and glutamic acid to aspartic acid) can be obtained by a simple two-step recombination (FIG. 5A). In the first recombination step, the PCR product with the gentamicin resistance gene and a counter selection marker (SacB) was transformed into P.aeruginosa cells by expressing the pBBR1-Rha-pluGam-TEpsy-kan plasmid or the pBBR1-Rha-RedGam-BAS-kan plasmid, and homologous recombination was performed with the target gene. Recombinant clones were randomly selected for colony PCR identification at the 3 'and 5' ends (fig. 5B), and the correct clones were subsequently functionally verified by SacB (fig. 6). In the second recombination step, a single-stranded DNA containing a point mutation (changing the codon at position 578 from GAG to GAC and changing the codon from glutamic acid to aspartic acid) was transformed into the previously constructed mutant by the plasmid pBBR1-Rha-pluGam-TEpsy-kan or pBBR1-Rha-RedGam-BAS-kan, and a point-mutated strain was obtained after recombination. The final mutant species were verified by colony PCR (fig. 5C) and sequencing (fig. 5D).

In order to expand the application range of the novel recombination system, the invention can also be used for expanding the application range of the novel recombination system by inserting a promoter (P)_apra) In order to activate the biosynthetic pathway of secondary metabolites. The promoter (P) was isolated by expression of pBBR1-Rha-BAS-kan plasmid_genta) Inserted into Pseudomonas syringae tomato variety DC3000(Pseudomonas syringae pv. tomato str. dc3000) (fig. 7A). However, none of the compounds was obtained in the HPLC-MS analysis.

In P.fluorescens, a mixed sensor kinase called RetS inhibits the production of several antibiotics, including 2, 4-diacetylphloroglucinol, pyrrolitro and pyoxanthin. The gene inactivation of retS in P.fluorescens may improve the biocontrol ability of this strain. The retS gene was knocked out by expression of the pBBR1-Rha-redGam-TEpsy-kan plasmid in P.fluorescens, and the mutant strain was verified by colony PCR (FIG. 7B).

Example 5: the expression of the SSB protein can improve the recombination efficiency of pseudomonas aeruginosa and escherichia coli

In E.coli, expression of SSB protein significantly improved the recombination efficiency of pBBR1-Rha-RedGam-BA-kan plasmid, but SSB protein did not improve the recombination efficiency of pBBR1-Rha-BA-kan plasmid (FIG. 8A), and similar results were obtained in P.aeruginosa (FIG. 8B). In P.syringae, the SSB protein could significantly improve the recombination efficiency of the pBBR1-Rha-BA-kan plasmid, but the recombination efficiency was rather decreased after the addition of Red. gamma.protein, and the SSB did not improve the recombination efficiency (FIG. 8C). The results indicate that SSB enhanced recombination efficiency may be correlated with host proteins.

Sequence listing

<110> Shandong university

Novel recombination system in <120> pseudomonas and application thereof

<141> 2017-04-18

<160> 30

<210> 1

<211> 84

<212> DNA

<213> Artificial sequence

<221> pluγ-TE-3

<222>（1）…（84）

<400> 1

acatgcaaat tttgatcgtg cgatacggac gcaacgtttc ttgcggacat atgatggcct 60

ccttcatata tcctccattt ttac 84

<210> 2

<211>73

<212> DNA

<213> Artificial sequence

<221> pluγ-TE-5

<222>（1）…（73）

<400> 2

gcgcttttta gactggtcgt aatgaacaat tcttaagaag gagatataca tatgaaccca 60

tatgcagttt atg 73

<210> 3

<211>84

<212> DNA

<213> Artificial sequence

<221> redγ-TE-3

<222>（1）…（84）

<400> 3

acatgcaaat tttgatcgtg cgatacggac gcaacgtttc ttgcggacat atgatggcct 60

cctttatacc tctgaatcaa tatc 84

<210> 4

<211> 72

<212> DNA

<213> Artificial sequence

<221> redγ-TE-5

<222>（1）…（72）

<400> 4

gcgcttttta gactggtcgt aatgaacaat tcttaagaag gagatataca tatggatatt 60

aatactgaaa ct 72

<210> 5

<211> 84

<212> DNA

<213> Artificial sequence

<221> pluγ-BAS-3

<222>（1）…（84）

<400> 5

tcgtagcgcg tggcgaactt cgtcaggagc ggtgttagtg cagttcccat cactagtcct 60

ccttcatata tcctccattt ttac 84

<210> 6

<211> 73

<212> DNA

<213> Artificial sequence

<221> pluγ-BAS-5

<222>（1）…（73）

<400> 6

gcgcttttta gactggtcgt aatgaacaat tcttaagaag gagatataca tatgaaccca 60

tatgcagttt atg 73

<210> 7

<211> 84

<212> DNA

<213> Artificial sequence

<221> redγ-BAS-3

<222>（1）…（84）

<400> 7

tcgtagcgcg tggcgaactt cgtcaggagc ggtgttagtg cagttcccat cactagtcct 60

cctttatacc tctgaatcaa tatc 84

<210> 8

<211> 72

<212> DNA

<213> Artificial sequence

<221> redγ-BAS-5

<222>（1）…（72）

<400> 8

gcgcttttta gactggtcgt aatgaacaat tcttaagaag gagatataca tatggatatt 60

aatactgaaa ct 72

<210> 9

<211> 85

<212> DNA

<213> Artificial sequence

<221> h-BAS-3

<222>（1）…（85）

<400> 9

tcgtagcgcg tggcgaactt cgtcaggagc ggtgttagtg cagttcccat cactagtcct 60

ccttcatttc gcccggctgc tgctg 85

<210> 10

<211> 72

<212> DNA

<213> Artificial sequence

<221> h-BAS-5

<222>（1）…（72）

<400> 10

gcgcttttta gactggtcgt aatgaacaat tcttaagaag gagatataca tatgatgact 60

accgagctga gc 72

<210> 11

<211> 98

<212> DNA

<213> Artificial sequence

<221> AB-genta-LoxM-3

<222>（1）…（98）

<400> 11

ttctccagca gacgctggcc gtgggtggtg agaatcgact cgggatggaa ctgcaagccc 60

agctgtcgat tgcgccaact taaatgtgaa agtgggtc 98

<210> 12

<211>97

<212> DNA

<213> Artificial sequence

<221> AB-genta-LoxM-5

<222>（1）…（97）

<400> 12

caattggggg aaaagggggg ttaccgatga tgaacatgcc gttgcgcgct agcgtcgcgc 60

aggccagtcg cccatagctg aattacattc ccaaccg 97

<210> 13

<211>96

<212> DNA

<213> Artificial sequence

<221> lasB-genta-loxM-3

<222>（1）…（96）

<400> 13

gaaggccttg cgggtatccc agcccggcga attggccaac aggtagaacg cacggttgta 60

cacgccgctg gagtgttagg tggcggtact tgggtc 96

<210> 14

<211> 98

<212> DNA

<213> Artificial sequence

<221> lasB-genta-loxM-5

<222>（1）…（98）

<400> 14

cgctacatgg accagcccag ccgcgacggg cgatccatcg acaacgcgtc gcagtactac 60

aacggcatcg acgtgataga ccagttgcaa tccaaacg 98

<210> 15

<211> 83

<212> DNA

<213> Artificial sequence

<221> mdoH-genta-loxM-3

<222>（1）…（83）

<400> 15

cctcttccca gacccgcgta tgcaggcgcg aaagtgctac cgggtcactc aacagagcca 60

caacttaaat gtgaaagtgg gtc 83

<210> 16

<211> 82

<212> DNA

<213> Artificial sequence

<221> mdoH-genta-loxM-5

<222>（1）…（82）

<400> 16

gacgaggcgc agaagccact gagcgaaacc tggagctatc agctgcctgc cgatgaataa 60

agctgaatta cattcccaac cg 82

<210> 17

<211> 98

<212> DNA

<213> Artificial sequence

<221> retS-genta-loxM-3

<222>（1）…（98）

<400> 17

tggagcatgg tgggagctca cgactaaagg agggcgagcg agagtttaac aggcgccgca 60

gagcctgtcg gctcacaact taaatgtgaa agtgggtc 98

<210> 18

<211> 97

<212> DNA

<213> Artificial sequence

<221> retS-genta-loxM-5

<222>（1）…（97）

<400> 18

gcacacgccc ttgccgtgcg gtcattacgc cgcgcatagt tataatcagg catcaaccaa 60

cgaagggatt tcgccagctg aattacattc ccaaccg 97

<210> 19

<211> 96

<212> DNA

<213> Artificial sequence

<221> toxA-sacB-genta-3

<222>（1）…（96）

<400> 19

gccgacgttg cgcgggtcgg tggggatcgc cgagggaatc accacggtgc gctcggccag 60

cggccagccg agaatttagg tggcggtact tgggtc 96

<210> 20

<211> 98

<212> DNA

<213> Artificial sequence

<221> toxA-sacB-genta-5

<222>（1）…（98）

<400> 20

acggctgatc ggccatccgc tgccgctgcg cctggacgcc atcaccggcc ccgaggagga 60

aggcgggcgc ctggaataga ccagttgcaa tccaaacg 98

<210> 21

<211> 96

<212> DNA

<213> Artificial sequence

<221> DC3000-BGC6-3

<222>（1）…（96）

<400> 21

tcgctacaac aagctggcca aaacctacgc agccatggtc acgctggtct gcagcctacg 60

ttgtagcggc gatacgaagg cacgaaccca gttgac 96

<210> 22

<211> 97

<212> DNA

<213> Artificial sequence

<221> DC3000-BGC6-5

<222>（1）…（97）

<400> 22

tacagaggac agttcatacg agaaatttga gtttttcgat gctttttgat ttgatggctt 60

catgcgtaaa catcctcgaa ttgttaggtg gcggtac 97

<210> 23

<211> 8261

<212> DNA

<213> Artificial sequence

<221> pBBR1-Rha-red_gba-kan

<222>（1）…（8261）

<400> 23

accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat cgggatatgc 60

aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg 120

aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca catttccccg 180

aaaagtgcca cctgggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag 240

cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt 300

tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa 360

gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc 420

aagatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 480

cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 540

aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 600

tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 660

gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 720

aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 780

tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 840

ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 900

ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 960

cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 1020

tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 1080

ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 1140

tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 1200

tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 1260

ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 1320

accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 1380

atcctccagc gcggggatct catgctggag ttcttcgccc acccccatgg gcaaatatta 1440

tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga 1500

tggcttccat gtcggcagaa tgcttaatga attacaacag tttttatgca ttaatctttc 1560

tgcgaattga gatgacgcca ctggctgggc gtcatcccgg tttcccgggt aaacaccacc 1620

gaaaaatagt tactatcttc aaagccacat tcggtcgaaa tatcactgat taacaggcgg 1680

ctatgctgga gaagatattg cgcatgacac actctgacct gtcgcagata ttgattgatg 1740

gtcattccag tctgctggcg aaattgctga cgcaaaacgc gctcactgca cgatgcctca 1800

tcacaaaatt tatccagcgc aaagggactt ttcaggctag ccgccagccg ggtaatcagc 1860

ttatccagca acgtttcgct ggatgttggc ggcaacgaat cactggtgta acgatggcga 1920

ttcagcaaca tcaccaactg cccgaacagc aactcagcca tttcgttagc aaacggcaca 1980

tgctgactac tttcatgctc aagctgaccg ataacctgcc gcgcctgcgc catccccatg 2040

ctacctaagc gccagtgtgg ttgccctgcg ctggcgttaa atcccggaat cgccccctgc 2100

cagtcaagat tcagcttcag acgctccggg caataaataa tattctgcaa aaccagatcg 2160

ttaacggaag cgtaggagtg tttatcgtca gcatgaatgt aaaagagatc gccacgggta 2220

atgcgataag ggcgatcgtt gagtacatgc aggccattac cgcgccagac aatcaccagc 2280

tcacaaaaat catgtgtatg ttcagcaaag acatcttgcg gataacggtc agccacagcg 2340

actgcctgct ggtcgctggc aaaaaaatca tctttgagaa gttttaactg atgcgccacc 2400

gtggctacct cggccagaga acgaagttga ttattcgcaa tatggcgtac aaatacgttg 2460

agaagattcg cgttattgca gaaagccatc ccgtccctgg cgaatatcac gcggtgacca 2520

gttaaactct cggcgaaaaa gcgtcgaaaa gtggttactg tcgctgaatc cacagcgata 2580

ggcgatgtca gtaacgctgg cctcgctgtg gcgtagcaga tgtcgggctt tcatcagtcg 2640

caggcggttc aggtatcgct gaggcgtcag tcccgtttgc tgcttaagct gccgatgtag 2700

cgtacgcagt gaaagagaaa attgatccgc cacggcatcc caattcacct catcggcaaa 2760

atggtcctcc agccaggcca gaagcaagtt gagacgtgat gcgctgtttt ccaggttctc 2820

ctgcaaactg cttttacgca gcaagagcag taattgcata aacaagatct cgcgactggc 2880

ggtcgagggt aaatcatttt ccccttcctg ctgttccatc tgtgcaacca gctgtcgcac 2940

ctgctgcaat acgctgtggt taacgcgcca gtgagacgga tactgcccat ccagctcttg 3000

tggcagcaac tgattcagcc cggcgagaaa ctgaaatcga tccggcgagc gatacagcac 3060

attggtcaga cacagattat cggtatgttc atacagatgc cgatcatgat cgcgtacgaa 3120

acagaccgtg ccaccggtga tggtataggg ctgcccatta aacacatgaa tacccgtgcc 3180

atgttcgaca atcacaattt catgaaaatc atgatgatgt tcaggaaaat ccgcctgcgg 3240

gagccggggt tctatcgcca cggacgcgtt accagacgga aaaaaatcca cactatgtaa 3300

tacggtcata ctggcctcct gatgtcgtca acacggcgaa atagtaatca cgaggtcagg 3360

ttcttacctt aaattttcga cggaaaacca cgtaaaaaac gtcgattttt caagatacag 3420

cgtgaatttt caggaaatgc ggtgagcatc acatcaccac aattcagcaa attgtgaaca 3480

tcatcacgtt catctttccc tggttgccaa tggcccattt tcctgtcagt aacgagaagg 3540

tcgcgaattc aggcgctttt tagactggtc gtaatgaaca attcttaaga aggagatata 3600

catatggata ttaatactga aactgagatc aagcaaaagc attcactaac cccctttcct 3660

gttttcctaa tcagcccggc atttcgcggg cgatattttc acagctattt caggagttca 3720

gccatgaacg cttattacat tcaggatcgt cttgaggctc agagctgggc gcgtcactac 3780

cagcagctcg cccgtgaaga gaaagaggca gaactggcag acgacatgga aaaaggcctg 3840

ccccagcacc tgtttgaatc gctatgcatc gatcatttgc aacgccacgg ggccagcaaa 3900

aaatccatta cccgtgcgtt tgatgacgat gttgagtttc aggagcgcat ggcagaacac 3960

atccggtaca tggttgaaac cattgctcac caccaggttg atattgattc agaggtataa 4020

aacgaatgag tactgcactc gcaacgctgg ctgggaagct ggctgaacgt gtcggcatgg 4080

attctgtcga cccacaggaa ctgatcacca ctcttcgcca gacggcattt aaaggtgatg 4140

ccagcgatgc gcagttcatc gcattactga tcgttgccaa ccagtacggc cttaatccgt 4200

ggacgaaaga aatttacgcc tttcctgata agcagaatgg catcgttccg gtggtgggcg 4260

ttgatggctg gtcccgcatc atcaatgaaa accagcagtt tgatggcatg gactttgagc 4320

aggacaatga atcctgtaca tgccggattt accgcaagga ccgtaatcat ccgatctgcg 4380

ttaccgaatg gatggatgaa tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa 4440

tcacggggcc gtggcagtcg catcccaaac ggatgttacg tcataaagcc atgattcagt 4500

gtgcccgtct ggccttcgga tttgctggta tctatgacaa ggatgaagcc gagcgcattg 4560

tcgaaaatac tgcatacact gcagaacgtc agccggaacg cgacatcact ccggttaacg 4620

atgaaaccat gcaggagatt aacactctgc tgatcgccct ggataaaaca tgggatgacg 4680

acttattgcc gctctgttcc cagatatttc gccgcgacat tcgtgcatcg tcagaactga 4740

cacaggccga agcagtaaaa gctcttggat tcctgaaaca gaaagccgca gagcagaagg 4800

tggcagcatg acaccggaca ttatcctgca gcgtaccggg atcgatgtga gagctgtcga 4860

acagggggat gatgcgtggc acaaattacg gctcggcgtc atcaccgctt cagaagttca 4920

caacgtgata gcaaaacccc gctccggaaa gaagtggcct gacatgaaaa tgtcctactt 4980

ccacaccctg cttgctgagg tttgcaccgg tgtggctccg gaagttaacg ctaaagcact 5040

ggcctgggga aaacagtacg agaacgacgc cagaaccctg tttgaattca cttccggcgt 5100

gaatgttact gaatccccga tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc 5160

cgatggttta tgcagtgacg gcaacggcct tgaactgaaa tgcccgttta cctcccggga 5220

tttcatgaag ttccggctcg gtggtttcga ggccataaag tcagcttaca tggcccaggt 5280

gcagtacagc atgtgggtga cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg 5340

tatgaagcgt gaaggcctgc attatgtcgt gattgagcgg gatgaaaagt acatggcgag 5400

ttttgacgag atcgtgccgg agttcatcga aaaaatggac gaggcactgg ctgaaattgg 5460

ttttgtattt ggggagcaat ggcgatagat gtacaagtaa gcttaccggt ttattgacta 5520

ccggaagcag tgtgaccgtg tgcttctcaa atgcctgagg ccagtttgct caggctctcc 5580

ccgtggaggt aataattgac gatatgatca tttattctgc ctcccagagc ctgataaaaa 5640

cggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg cccggctcca 5700

tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgaggc ggctacagcc 5760

gatagtctgg aacagcgcac ttacgggttg ctgcgcaacc caagtgctac cggcgcggca 5820

gcgtgacccg tgtcggcggc tccaacggct cgccatcgtc cagaaaacac ggctcatcgg 5880

gcatcggcag gcgctgctgc ccgcgccgtt cccattcctc cgtttcggtc aaggctggca 5940

ggtctggttc catgcccgga atgccgggct ggctgggcgg ctcctcgccg gggccggtcg 6000

gtagttgctg ctcgcccgga tacagggtcg ggatgcggcg caggtcgcca tgccccaaca 6060

gcgattcgtc ctggtcgtcg tgatcaacca ccacggcggc actgaacacc gacaggcgca 6120

actggtcgcg gggctggccc cacgccacgc ggtcattgac cacgtaggcc gacacggtgc 6180

cggggccgtt gagcttcacg acggagatcc agcgctcggc caccaagtcc ttgactgcgt 6240

attggaccgt ccgcaaagaa cgtccgatga gcttggaaag tgtcttctgg ctgaccacca 6300

cggcgttctg gtggcccatc tgcgccacga ggtgatgcag cagcattgcc gccgtgggtt 6360

tcctcgcaat aagcccggcc cacgcctcat gcgctttgcg ttccgtttgc acccagtgac 6420

cgggcttgtt cttggcttga atgccgattt ctctggactg cgtggccatg cttatctcca 6480

tgcggtaggg tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact tttcggcagc 6540

gcgacaacaa ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc tttaacctac 6600

gcaatgagct attgcggggg gtgccgcaat gagctgttgc gtacccccct tttttaagtt 6660

gttgattttt aagtctttcg catttcgccc tatatctagt tctttggtgc ccaaagaagg 6720

gcacccctgc ggggttcccc cacgccttcg gcgcggctcc ccctccggca aaaagtggcc 6780

cctccggggc ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg gcgctgcccc 6840

cttggaaccc ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg gcttcgcctt 6900

cgactgcccc cactcgcata ggcttgggtc gttccaggcg cgtcaaggcc aagccgctgc 6960

gcggtcgctg cgcgagcctt gacccgcctt ccacttggtg tccaaccggc aagcgaagcg 7020

cgcaggccgc aggccggagg cttttcccca gagaaaatta aaaaaattga tggggcaagg 7080

ccgcaggccg cgcagttgga gccggtgggt atgtggtcga aggctgggta gccggtgggc 7140

aatccctgtg gtcaagctcg tgggcaggcg cagcctgtcc atcagcttgt ccagcagggt 7200

tgtccacggg ccgagcgaag cgagccagcc ggtggccgct cgcggccatc gtccacatat 7260

ccacgggctg gcaagggagc gcagcgaccg cgcagggcga agcccggaga gcaagcccgt 7320

agggcgccgc agccgccgta ggcggtcacg actttgcgaa gcaaagtcta gtgagtatac 7380

tcaagcattg agtggcccgc cggaggcacc gccttgcgct gcccccgtcg agccggttgg 7440

acaccaaaag ggaggggcag gcatggcggc atacgcgatc atgcgatgca agaagctggc 7500

gaaaatgggc aacgtggcgg ccagtctcaa gcacgcctac cgcgagcgcg agacgcccaa 7560

cgctgacgcc agcaggacgc cagagaacga gcactgggcg gccagcagca ccgatgaagc 7620

gatgggccga ctgcgcgagt tgctgccaga gaagcggcgc aaggacgctg tgttggcggt 7680

cgagtacgtc atgacggcca gcccggaatg gtggaagtcg gccagccaag aacagcaggc 7740

ggcgttcttc gagaaggcgc acaagtggct ggcggacaag tacggggcgg atcgcatcgt 7800

gacggccagc atccaccgtg acgaaaccag cccgcacatg accgcgttcg tggtgccgct 7860

gacgcaggac ggcaggctgt cggccaagga gttcatcggc aacaaagcgc agatgacccg 7920

cgaccagacc acgtttgcgg ccgctgtggc cgatctaggg ctgcaacggg gcatcgaggg 7980

cagcaaggca cgtcacacgc gcattcaggc gttctacgag gccctggagc ggccaccagt 8040

gggccacgtc accatcagcc cgcaagcggt cgagccacgc gcctatgcac cgcagggatt 8100

ggccgaaaag ctgggaatct caaagcgcgt tgagacgccg gaagccgtgg ccgaccggct 8160

gacaaaagcg gttcggcagg ggtatgagcc tgccctacag gccgccgcag gagcgcgtga 8220

gatgcgcaag aaggccgatc aagcccaaga gacggcccga g 8261

<210> 24

<211> 8240

<212> DNA

<213> Artificial sequence

<221> pBBR1-Rha-plu_gba-kan

<222>（1）…（8240）

<400> 24

accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat cgggatatgc 60

aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg 120

aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca catttccccg 180

aaaagtgcca cctgggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag 240

cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt 300

tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa 360

gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc 420

aagatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 480

cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 540

aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 600

tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 660

gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 720

aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 780

tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 840

ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 900

ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 960

cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 1020

tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 1080

ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 1140

tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 1200

tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 1260

ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 1320

accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 1380

atcctccagc gcggggatct catgctggag ttcttcgccc acccccatgg gcaaatatta 1440

tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga 1500

tggcttccat gtcggcagaa tgcttaatga attacaacag tttttatgca ttaatctttc 1560

tgcgaattga gatgacgcca ctggctgggc gtcatcccgg tttcccgggt aaacaccacc 1620

gaaaaatagt tactatcttc aaagccacat tcggtcgaaa tatcactgat taacaggcgg 1680

ctatgctgga gaagatattg cgcatgacac actctgacct gtcgcagata ttgattgatg 1740

gtcattccag tctgctggcg aaattgctga cgcaaaacgc gctcactgca cgatgcctca 1800

tcacaaaatt tatccagcgc aaagggactt ttcaggctag ccgccagccg ggtaatcagc 1860

ttatccagca acgtttcgct ggatgttggc ggcaacgaat cactggtgta acgatggcga 1920

ttcagcaaca tcaccaactg cccgaacagc aactcagcca tttcgttagc aaacggcaca 1980

tgctgactac tttcatgctc aagctgaccg ataacctgcc gcgcctgcgc catccccatg 2040

ctacctaagc gccagtgtgg ttgccctgcg ctggcgttaa atcccggaat cgccccctgc 2100

cagtcaagat tcagcttcag acgctccggg caataaataa tattctgcaa aaccagatcg 2160

ttaacggaag cgtaggagtg tttatcgtca gcatgaatgt aaaagagatc gccacgggta 2220

atgcgataag ggcgatcgtt gagtacatgc aggccattac cgcgccagac aatcaccagc 2280

tcacaaaaat catgtgtatg ttcagcaaag acatcttgcg gataacggtc agccacagcg 2340

actgcctgct ggtcgctggc aaaaaaatca tctttgagaa gttttaactg atgcgccacc 2400

gtggctacct cggccagaga acgaagttga ttattcgcaa tatggcgtac aaatacgttg 2460

agaagattcg cgttattgca gaaagccatc ccgtccctgg cgaatatcac gcggtgacca 2520

gttaaactct cggcgaaaaa gcgtcgaaaa gtggttactg tcgctgaatc cacagcgata 2580

ggcgatgtca gtaacgctgg cctcgctgtg gcgtagcaga tgtcgggctt tcatcagtcg 2640

caggcggttc aggtatcgct gaggcgtcag tcccgtttgc tgcttaagct gccgatgtag 2700

cgtacgcagt gaaagagaaa attgatccgc cacggcatcc caattcacct catcggcaaa 2760

atggtcctcc agccaggcca gaagcaagtt gagacgtgat gcgctgtttt ccaggttctc 2820

ctgcaaactg cttttacgca gcaagagcag taattgcata aacaagatct cgcgactggc 2880

ggtcgagggt aaatcatttt ccccttcctg ctgttccatc tgtgcaacca gctgtcgcac 2940

ctgctgcaat acgctgtggt taacgcgcca gtgagacgga tactgcccat ccagctcttg 3000

tggcagcaac tgattcagcc cggcgagaaa ctgaaatcga tccggcgagc gatacagcac 3060

attggtcaga cacagattat cggtatgttc atacagatgc cgatcatgat cgcgtacgaa 3120

acagaccgtg ccaccggtga tggtataggg ctgcccatta aacacatgaa tacccgtgcc 3180

atgttcgaca atcacaattt catgaaaatc atgatgatgt tcaggaaaat ccgcctgcgg 3240

gagccggggt tctatcgcca cggacgcgtt accagacgga aaaaaatcca cactatgtaa 3300

tacggtcata ctggcctcct gatgtcgtca acacggcgaa atagtaatca cgaggtcagg 3360

ttcttacctt aaattttcga cggaaaacca cgtaaaaaac gtcgattttt caagatacag 3420

cgtgaatttt caggaaatgc ggtgagcatc acatcaccac aattcagcaa attgtgaaca 3480

tcatcacgtt catctttccc tggttgccaa tggcccattt tcctgtcagt aacgagaagg 3540

tcgcgaattc aggcgctttt tagactggtc gtaatgaaca attcttaaga aggagatact 3600

agtatgaacc catatgcagt ttatgatgag attgaagaaa agaggttgga agatgagcac 3660

tacagggaaa ttatattaga gcaacaaggc atggatgccg aaattattta taacaagctt 3720

cctgagcttg ctggaatttt ttcaatcgaa actaacaaac tctttggcga attactcacg 3780

gagaacgatg aagccgcaga gctcgttaat tctcttttgt atgaactaag cttaatgaaa 3840

gtaaaaatgg aggatatatg agcacagcag tacaaaaagt ttatgaaatc atcaacccgc 3900

tcaagactga atttgagcaa atttgtagtg agccgggcat agtatttaaa cgtgagtctg 3960

aatttgctat gcagatattc gcaaataatg attttttagc cacaactgca ttaaataatc 4020

cggtttcagc gcgtagcgca gtaatgaaca ttgcagcaat tggcattagt ctaaaccctg 4080

cgcaaaagct ggcttatcta gtgccacgaa acaaaagcgt gtgtcttgat atcagttaca 4140

tgggtttgat gcacatcgct caacaatcgg gtgctattaa gtggtgtcaa tccgctgttg 4200

ttcgaaaaaa tgacatattc aagcgcacat ctattgatac tgcgccaatt cacgaataca 4260

acgaattcga cacagcgcaa tcgagggggg acattgtcgg cgcgtataca gtaatcaaaa 4320

cggatgactg tgattacatt actcacacaa tgagagcatc agctatattt gatatcagag 4380

atagatcatc ggcatggata gcatataaaa caaaagggaa atcgtgtccg tgggtcacgg 4440

acgaggaaca aatgatttta aaaaccgtag tcaagcaggc ggctaagtat tggcctcgca 4500

gggagcgatt agataaagcc attgactatg taaacacaga ggcgggtgaa ggaattgatt 4560

ttagcaaagg tcaaaactct tacattgacg taactcccgc agcagatagc acaatcgaaa 4620

gtataacaaa cttacttaca acaatgaata aaacttggga cgatgattta cttccacttt 4680

gttcaaagat attcagacga caaatattat cacccgccga attaacagag tcggaggcaa 4740

ttaaagcatc tgatttttta aggaaaaagg catcataaat gatcagcaat gaactcattc 4800

tcagcaaaac aggggttgat ttgctcacag tcgaacaggg aagtcatgat tggcagcggt 4860

taagattggg agtaataacg gcatcggaag ccggaaaagt gatatcaaaa ccgcgtagtg 4920

gtacaaaatg gacagacatg aagctgacat atttctatac acttctgggc gaagtgtgca 4980

caggtagtac gccggaagta aatgcaaaaa cattagcgtg gggaaaaaat tacgaagaat 5040

cagcaagagc aacttttgaa tttgtagctg atgtaaatgt aacggaaact tcaattctat 5100

ttaaagatga atcgctcaga accgcatgct caccagatgg tatttgtagt gacggacttg 5160

ggcttgaact taaatgtcct tttactactg ctgtatttat gaaattccgc ttgggtggtt 5220

ttgatgctat taaatctgaa tatatggctc aagttcaata ttcgatgtgg gtcacaggta 5280

aaaatgaatg gtactttgct aattatgacc cgcgtatgac tcgtgaaggg attcattacg 5340

ttgttgttga gcgtgacaac gaatatatga gtcagtttaa tgatatggtt cccgatttca 5400

tagaaaaaat ggacgaatca ttaaatgaaa tcggcttcaa atttggcgat caatggaaat 5460

gaggatcctg tacaagtaag cttaccggtt tattgactac cggaagcagt gtgaccgtgt 5520

gcttctcaaa tgcctgaggc cagtttgctc aggctctccc cgtggaggta ataattgacg 5580

atatgatcat ttattctgcc tcccagagcc tgataaaaac ggtgaatccg ttagcgaggt 5640

gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg 5700

gaggcagaca aggtataggg cggcgaggcg gctacagccg atagtctgga acagcgcact 5760

tacgggttgc tgcgcaaccc aagtgctacc ggcgcggcag cgtgacccgt gtcggcggct 5820

ccaacggctc gccatcgtcc agaaaacacg gctcatcggg catcggcagg cgctgctgcc 5880

cgcgccgttc ccattcctcc gtttcggtca aggctggcag gtctggttcc atgcccggaa 5940

tgccgggctg gctgggcggc tcctcgccgg ggccggtcgg tagttgctgc tcgcccggat 6000

acagggtcgg gatgcggcgc aggtcgccat gccccaacag cgattcgtcc tggtcgtcgt 6060

gatcaaccac cacggcggca ctgaacaccg acaggcgcaa ctggtcgcgg ggctggcccc 6120

acgccacgcg gtcattgacc acgtaggccg acacggtgcc ggggccgttg agcttcacga 6180

cggagatcca gcgctcggcc accaagtcct tgactgcgta ttggaccgtc cgcaaagaac 6240

gtccgatgag cttggaaagt gtcttctggc tgaccaccac ggcgttctgg tggcccatct 6300

gcgccacgag gtgatgcagc agcattgccg ccgtgggttt cctcgcaata agcccggccc 6360

acgcctcatg cgctttgcgt tccgtttgca cccagtgacc gggcttgttc ttggcttgaa 6420

tgccgatttc tctggactgc gtggccatgc ttatctccat gcggtagggt gccgcacggt 6480

tgcggcacca tgcgcaatca gctgcaactt ttcggcagcg cgacaacaat tatgcgttgc 6540

gtaaaagtgg cagtcaatta cagattttct ttaacctacg caatgagcta ttgcgggggg 6600

tgccgcaatg agctgttgcg tacccccctt ttttaagttg ttgattttta agtctttcgc 6660

atttcgccct atatctagtt ctttggtgcc caaagaaggg cacccctgcg gggttccccc 6720

acgccttcgg cgcggctccc cctccggcaa aaagtggccc ctccggggct tgttgatcga 6780

ctgcgcggcc ttcggccttg cccaaggtgg cgctgccccc ttggaacccc cgcactcgcc 6840

gccgtgaggc tcggggggca ggcgggcggg cttcgccttc gactgccccc actcgcatag 6900

gcttgggtcg ttccaggcgc gtcaaggcca agccgctgcg cggtcgctgc gcgagccttg 6960

acccgccttc cacttggtgt ccaaccggca agcgaagcgc gcaggccgca ggccggaggc 7020

ttttccccag agaaaattaa aaaaattgat ggggcaaggc cgcaggccgc gcagttggag 7080

ccggtgggta tgtggtcgaa ggctgggtag ccggtgggca atccctgtgg tcaagctcgt 7140

gggcaggcgc agcctgtcca tcagcttgtc cagcagggtt gtccacgggc cgagcgaagc 7200

gagccagccg gtggccgctc gcggccatcg tccacatatc cacgggctgg caagggagcg 7260

cagcgaccgc gcagggcgaa gcccggagag caagcccgta gggcgccgca gccgccgtag 7320

gcggtcacga ctttgcgaag caaagtctag tgagtatact caagcattga gtggcccgcc 7380

ggaggcaccg ccttgcgctg cccccgtcga gccggttgga caccaaaagg gaggggcagg 7440

catggcggca tacgcgatca tgcgatgcaa gaagctggcg aaaatgggca acgtggcggc 7500

cagtctcaag cacgcctacc gcgagcgcga gacgcccaac gctgacgcca gcaggacgcc 7560

agagaacgag cactgggcgg ccagcagcac cgatgaagcg atgggccgac tgcgcgagtt 7620

gctgccagag aagcggcgca aggacgctgt gttggcggtc gagtacgtca tgacggccag 7680

cccggaatgg tggaagtcgg ccagccaaga acagcaggcg gcgttcttcg agaaggcgca 7740

caagtggctg gcggacaagt acggggcgga tcgcatcgtg acggccagca tccaccgtga 7800

cgaaaccagc ccgcacatga ccgcgttcgt ggtgccgctg acgcaggacg gcaggctgtc 7860

ggccaaggag ttcatcggca acaaagcgca gatgacccgc gaccagacca cgtttgcggc 7920

cgctgtggcc gatctagggc tgcaacgggg catcgagggc agcaaggcac gtcacacgcg 7980

cattcaggcg ttctacgagg ccctggagcg gccaccagtg ggccacgtca ccatcagccc 8040

gcaagcggtc gagccacgcg cctatgcacc gcagggattg gccgaaaagc tgggaatctc 8100

aaagcgcgtt gagacgccgg aagccgtggc cgaccggctg acaaaagcgg ttcggcaggg 8160

gtatgagcct gccctacagg ccgccgcagg agcgcgtgag atgcgcaaga aggccgatca 8220

agcccaagag acggcccgag 8240

<210> 25

<211> 8921

<212> DNA

<213> Artificial sequence

<221> pBBR1-Rha-TEpsy-kan

<222>（1）…（8921）

<400> 25

atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgggcaa cgtggcggcc 60

agtctcaagc acgcctaccg cgagcgcgag acgcccaacg ctgacgccag caggacgcca 120

gagaacgagc actgggcggc cagcagcacc gatgaagcga tgggccgact gcgcgagttg 180

ctgccagaga agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat gacggccagc 240

ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga gaaggcgcac 300

aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat ccaccgtgac 360

gaaaccagcc cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg caggctgtcg 420

gccaaggagt tcatcggcaa caaagcgcag atgacccgcg accagaccac gtttgcggcc 480

gctgtggccg atctagggct gcaacggggc atcgagggca gcaaggcacg tcacacgcgc 540

attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac catcagcccg 600

caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct gggaatctca 660

aagcgcgttg agacgccgga agccgtggcc gaccggctga caaaagcggt tcggcagggg 720

tatgagcctg ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa ggccgatcaa 780

gcccaagaga cggcccgaga ccttcgggag cgcctgaagc ccgttctgga cgccctgggg 840

ccgttgaatc gggatatgca ggccaaggcc gccgcgatca tcaaggccgt gggcgaaaag 900

ctgctgacgg aacagcggga agtccagcgc cagaaacagg cccagcgcca gcaggaacgc 960

gggcgcgcac atttccccga aaagtgccac ctgggatgaa tgtcagctac tgggctatct 1020

ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc 1080

gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc 1140

cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga 1200

tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga 1260

ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 1320

atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 1380

aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 1440

acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 1500

acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 1560

tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 1620

ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 1680

agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 1740

atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 1800

aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 1860

gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 1920

cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 1980

tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 2040

agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 2100

atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 2160

ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 2220

cccccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg 2280

ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt 2340

ttttatgcat taatctttct gcgaattgag atgacgccac tggctgggcg tcatcccggt 2400

ttcccgggta aacaccaccg aaaaatagtt actatcttca aagccacatt cggtcgaaat 2460

atcactgatt aacaggcggc tatgctggag aagatattgc gcatgacaca ctctgacctg 2520

tcgcagatat tgattgatgg tcattccagt ctgctggcga aattgctgac gcaaaacgcg 2580

ctcactgcac gatgcctcat cacaaaattt atccagcgca aagggacttt tcaggctagc 2640

cgccagccgg gtaatcagct tatccagcaa cgtttcgctg gatgttggcg gcaacgaatc 2700

actggtgtaa cgatggcgat tcagcaacat caccaactgc ccgaacagca actcagccat 2760

ttcgttagca aacggcacat gctgactact ttcatgctca agctgaccga taacctgccg 2820

cgcctgcgcc atccccatgc tacctaagcg ccagtgtggt tgccctgcgc tggcgttaaa 2880

tcccggaatc gccccctgcc agtcaagatt cagcttcaga cgctccgggc aataaataat 2940

attctgcaaa accagatcgt taacggaagc gtaggagtgt ttatcgtcag catgaatgta 3000

aaagagatcg ccacgggtaa tgcgataagg gcgatcgttg agtacatgca ggccattacc 3060

gcgccagaca atcaccagct cacaaaaatc atgtgtatgt tcagcaaaga catcttgcgg 3120

ataacggtca gccacagcga ctgcctgctg gtcgctggca aaaaaatcat ctttgagaag 3180

ttttaactga tgcgccaccg tggctacctc ggccagagaa cgaagttgat tattcgcaat 3240

atggcgtaca aatacgttga gaagattcgc gttattgcag aaagccatcc cgtccctggc 3300

gaatatcacg cggtgaccag ttaaactctc ggcgaaaaag cgtcgaaaag tggttactgt 3360

cgctgaatcc acagcgatag gcgatgtcag taacgctggc ctcgctgtgg cgtagcagat 3420

gtcgggcttt catcagtcgc aggcggttca ggtatcgctg aggcgtcagt cccgtttgct 3480

gcttaagctg ccgatgtagc gtacgcagtg aaagagaaaa ttgatccgcc acggcatccc 3540

aattcacctc atcggcaaaa tggtcctcca gccaggccag aagcaagttg agacgtgatg 3600

cgctgttttc caggttctcc tgcaaactgc ttttacgcag caagagcagt aattgcataa 3660

acaagatctc gcgactggcg gtcgagggta aatcattttc cccttcctgc tgttccatct 3720

gtgcaaccag ctgtcgcacc tgctgcaata cgctgtggtt aacgcgccag tgagacggat 3780

actgcccatc cagctcttgt ggcagcaact gattcagccc ggcgagaaac tgaaatcgat 3840

ccggcgagcg atacagcaca ttggtcagac acagattatc ggtatgttca tacagatgcc 3900

gatcatgatc gcgtacgaaa cagaccgtgc caccggtgat ggtatagggc tgcccattaa 3960

acacatgaat acccgtgcca tgttcgacaa tcacaatttc atgaaaatca tgatgatgtt 4020

caggaaaatc cgcctgcggg agccggggtt ctatcgccac ggacgcgtta ccagacggaa 4080

aaaaatccac actatgtaat acggtcatac tggcctcctg atgtcgtcaa cacggcgaaa 4140

tagtaatcac gaggtcaggt tcttacctta aattttcgac ggaaaaccac gtaaaaaacg 4200

tcgatttttc aagatacagc gtgaattttc aggaaatgcg gtgagcatca catcaccaca 4260

attcagcaaa ttgtgaacat catcacgttc atctttccct ggttgccaat ggcccatttt 4320

cctgtcagta acgagaaggt cgcgaattca ggcgcttttt agactggtcg taatgaacaa 4380

ttcttaagaa ggagatatac atatgtccgc aagaaacgtt gcgtccgtat cgcacgatca 4440

aaatttgcat gtgcttccgc atgcggccac ctcgaccagc gccttggtcc tagacggcga 4500

cagcctggac aagatgatgc gcctggccga ggtcatggcg accgggcgag caactctgcc 4560

gaagcacttc aacggcaact ccgccgattg cctggccgtc gtcatgcagg ccatgcagtg 4620

gaggatgaac ccgtttgccg tcgcacagaa aacccatctg gtgaatggcg tgctcggtta 4680

cgaggcgcag ctggtcaacg ccgtgattac aacctgcgca cctgtcgtgg atcgcttgca 4740

ctacgagtgg tacggcaact gggaaaaggt catcggcaag tttgtgatca aaaacagcga 4800

caagggtgag taccggcagc caggctggaa gcttgccgac gaggaaggcc tgggcgtgaa 4860

ggtttgggcg acattccgcg gagaagacga gcccagggtg ttggagctgc tactggccca 4920

ggcgcgtaca cgcaacagca cgctctgggc cgacgatcct cgccagcagc tcgcgtacct 4980

ggcaaccaaa cgctggtccc gcctctactg cccggacgtg attctggggg tttacagccc 5040

agatgagctg gaggaaagca cccctcggta ccgtgatgtt tccccacagc cggagcagca 5100

agggtccgag cttcccgcct acgaggacga aaagttcaaa acgatgtttc ccaaatggca 5160

ggagggaatc gacaccggaa agaccgacac cgaaagcctg atcgccttct tggaatcgaa 5220

atacaccctc agtgccgatc agatcgaccg aatcaaccag atggctccta tcgccggaga 5280

aaccgcatga agacgcataa agtcacccag ggcagtgccg aatggcacgc gctccgcgcc 5340

aactatcgca ccgcgtctga agcaccggtt atgatgggcg catccaagca gatgaagcgc 5400

accgagctgc ttcatgccaa gaagaccggg ctcgaccggg acatttcgtg gtgggtccag 5460

aagtacttgt tcgacaaggg tcacgaagcc gaagcgctgg cgcgaccaat tctcgaaggt 5520

cgaatcggcg aagacctgtt cccggttgtg ggcaccgaag gtgacctgct ggcctccctc 5580

gacggctgca caatgatggg cgagatcgtt ttcgagcaca aaatgtggaa cgagcagctc 5640

gccgccgacg tgcgtgcggg cgatctggat gcgcattact actggcagct ggagcagcaa 5700

ctgctggtgt caggtgctga gaaagtgatt ttcgtttgct cggacgggac cgaagacaac 5760

ttcgtctcga tggagtactt cccggtgaac ggtcgggcgg cgaaactggt cgccggctgg 5820

aaacagttcg aagtcgacct tgaagcgtat gagccaactg aggccgtggt ggaggcagtt 5880

gggaagactc cggacgcgct gccagcgctg cgcatcgaag taaccggaat ggtcactgcg 5940

agcaacctgg agcagttcaa ggcccattcg ctggccgtgt tttcggcgat caacactgac 6000

ctgcagaccg accagcactt cgctgatgca gagaaaacag tgaagtggtg tggcgaggtc 6060

gaggagcgcc tggaagcagc caagcaacat gcgctgagcc agacggaaac catcgacgcc 6120

ctcttccgca ccatcgacga aatatcggag caggcccgtc gcaaacggct tgagctggac 6180

aaactggtga aagctcgcaa ggtggccatt cgcgaagaga tcgttctgaa agcgaaagca 6240

gcgctgcgtg atcatctgga caagatcaac acctcgttcg gcggcaaggt gcggctgccg 6300

gagatccccg cggacttcgc cggcgcgatc aaaggcaaaa aaaatatcgc cagtctgcgt 6360

gacgccgccg actctgaact ggcccgggcc aagatcgagg ccagccagat cggcgacggt 6420

atccgcgcca acttggaaag cttgcgctcg ctcgccgtgg atcatgcgtt tctgttcaac 6480

gatgctcagc agatcgtgct caagaacaat gacgacctgg tggccctcat caaggtgcgg 6540

atcaacgagc acaagcaggc cgaggaagcg aaagagctgg cgcagcgtga acgtattcgt 6600

gctgaagaat cagcgaagct tgcggctgcc gccgaagcgg aacaagttgc cgaagcggaa 6660

aaggcaacag ccaacgcgcc cgcgccgcaa gctgctgagg catccaaacc agtcgaacaa 6720

ccagctccga gaatgtcggc cgtcacccca tctgcgaagg tgccgcccaa gcctacaaag 6780

ctcgaagcga acgtcaccga ccttcatgcg ctggtaaaag ccgtctacga aggtcgtgcg 6840

ccgatatcgg tcctcacagt gaactggggc gcgctggacg accttgtcca cattcaaggc 6900

gctgacttcc aaatggacgg agtaaccatc acgcaggtgg cagcatgatg tacaagtaag 6960

cttaccggtt tattgactac cggaagcagt gtgaccgtgt gcttctcaaa tgcctgaggc 7020

cagtttgctc aggctctccc cgtggaggta ataattgacg atatgatcat ttattctgcc 7080

tcccagagcc tgataaaaac ggtgaatccg ttagcgaggt gccgccggct tccattcagg 7140

tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg 7200

cggcgaggcg gctacagccg atagtctgga acagcgcact tacgggttgc tgcgcaaccc 7260

aagtgctacc ggcgcggcag cgtgacccgt gtcggcggct ccaacggctc gccatcgtcc 7320

agaaaacacg gctcatcggg catcggcagg cgctgctgcc cgcgccgttc ccattcctcc 7380

gtttcggtca aggctggcag gtctggttcc atgcccggaa tgccgggctg gctgggcggc 7440

tcctcgccgg ggccggtcgg tagttgctgc tcgcccggat acagggtcgg gatgcggcgc 7500

aggtcgccat gccccaacag cgattcgtcc tggtcgtcgt gatcaaccac cacggcggca 7560

ctgaacaccg acaggcgcaa ctggtcgcgg ggctggcccc acgccacgcg gtcattgacc 7620

acgtaggccg acacggtgcc ggggccgttg agcttcacga cggagatcca gcgctcggcc 7680

accaagtcct tgactgcgta ttggaccgtc cgcaaagaac gtccgatgag cttggaaagt 7740

gtcttctggc tgaccaccac ggcgttctgg tggcccatct gcgccacgag gtgatgcagc 7800

agcattgccg ccgtgggttt cctcgcaata agcccggccc acgcctcatg cgctttgcgt 7860

tccgtttgca cccagtgacc gggcttgttc ttggcttgaa tgccgatttc tctggactgc 7920

gtggccatgc ttatctccat gcggtagggt gccgcacggt tgcggcacca tgcgcaatca 7980

gctgcaactt ttcggcagcg cgacaacaat tatgcgttgc gtaaaagtgg cagtcaatta 8040

cagattttct ttaacctacg caatgagcta ttgcgggggg tgccgcaatg agctgttgcg 8100

tacccccctt ttttaagttg ttgattttta agtctttcgc atttcgccct atatctagtt 8160

ctttggtgcc caaagaaggg cacccctgcg gggttccccc acgccttcgg cgcggctccc 8220

cctccggcaa aaagtggccc ctccggggct tgttgatcga ctgcgcggcc ttcggccttg 8280

cccaaggtgg cgctgccccc ttggaacccc cgcactcgcc gccgtgaggc tcggggggca 8340

ggcgggcggg cttcgccttc gactgccccc actcgcatag gcttgggtcg ttccaggcgc 8400

gtcaaggcca agccgctgcg cggtcgctgc gcgagccttg acccgccttc cacttggtgt 8460

ccaaccggca agcgaagcgc gcaggccgca ggccggaggc ttttccccag agaaaattaa 8520

aaaaattgat ggggcaaggc cgcaggccgc gcagttggag ccggtgggta tgtggtcgaa 8580

ggctgggtag ccggtgggca atccctgtgg tcaagctcgt gggcaggcgc agcctgtcca 8640

tcagcttgtc cagcagggtt gtccacgggc cgagcgaagc gagccagccg gtggccgctc 8700

gcggccatcg tccacatatc cacgggctgg caagggagcg cagcgaccgc gcagggcgaa 8760

gcccggagag caagcccgta gggcgccgca gccgccgtag gcggtcacga ctttgcgaag 8820

caaagtctag tgagtatact caagcattga gtggcccgcc ggaggcaccg ccttgcgctg 8880

cccccgtcga gccggttgga caccaaaagg gaggggcagg c 8921

<210> 26

<211>9192

<212> DNA

<213> Artificial sequence

<221> pBBR1-Rha-pluGam-TEpsy-kan

<222>（1）…（9192）

<400> 26

accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat cgggatatgc 60

aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg 120

aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca catttccccg 180

aaaagtgcca cctgggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag 240

cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt 300

tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa 360

gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc 420

aagatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 480

cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 540

aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 600

tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 660

gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 720

aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 780

tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 840

ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 900

ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 960

cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 1020

tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 1080

ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 1140

tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 1200

tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 1260

ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 1320

accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 1380

atcctccagc gcggggatct catgctggag ttcttcgccc acccccatgg gcaaatatta 1440

tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga 1500

tggcttccat gtcggcagaa tgcttaatga attacaacag tttttatgca ttaatctttc 1560

tgcgaattga gatgacgcca ctggctgggc gtcatcccgg tttcccgggt aaacaccacc 1620

gaaaaatagt tactatcttc aaagccacat tcggtcgaaa tatcactgat taacaggcgg 1680

ctatgctgga gaagatattg cgcatgacac actctgacct gtcgcagata ttgattgatg 1740

gtcattccag tctgctggcg aaattgctga cgcaaaacgc gctcactgca cgatgcctca 1800

tcacaaaatt tatccagcgc aaagggactt ttcaggctag ccgccagccg ggtaatcagc 1860

ttatccagca acgtttcgct ggatgttggc ggcaacgaat cactggtgta acgatggcga 1920

ttcagcaaca tcaccaactg cccgaacagc aactcagcca tttcgttagc aaacggcaca 1980

tgctgactac tttcatgctc aagctgaccg ataacctgcc gcgcctgcgc catccccatg 2040

ctacctaagc gccagtgtgg ttgccctgcg ctggcgttaa atcccggaat cgccccctgc 2100

cagtcaagat tcagcttcag acgctccggg caataaataa tattctgcaa aaccagatcg 2160

ttaacggaag cgtaggagtg tttatcgtca gcatgaatgt aaaagagatc gccacgggta 2220

atgcgataag ggcgatcgtt gagtacatgc aggccattac cgcgccagac aatcaccagc 2280

tcacaaaaat catgtgtatg ttcagcaaag acatcttgcg gataacggtc agccacagcg 2340

actgcctgct ggtcgctggc aaaaaaatca tctttgagaa gttttaactg atgcgccacc 2400

gtggctacct cggccagaga acgaagttga ttattcgcaa tatggcgtac aaatacgttg 2460

agaagattcg cgttattgca gaaagccatc ccgtccctgg cgaatatcac gcggtgacca 2520

gttaaactct cggcgaaaaa gcgtcgaaaa gtggttactg tcgctgaatc cacagcgata 2580

ggcgatgtca gtaacgctgg cctcgctgtg gcgtagcaga tgtcgggctt tcatcagtcg 2640

caggcggttc aggtatcgct gaggcgtcag tcccgtttgc tgcttaagct gccgatgtag 2700

cgtacgcagt gaaagagaaa attgatccgc cacggcatcc caattcacct catcggcaaa 2760

atggtcctcc agccaggcca gaagcaagtt gagacgtgat gcgctgtttt ccaggttctc 2820

ctgcaaactg cttttacgca gcaagagcag taattgcata aacaagatct cgcgactggc 2880

ggtcgagggt aaatcatttt ccccttcctg ctgttccatc tgtgcaacca gctgtcgcac 2940

ctgctgcaat acgctgtggt taacgcgcca gtgagacgga tactgcccat ccagctcttg 3000

tggcagcaac tgattcagcc cggcgagaaa ctgaaatcga tccggcgagc gatacagcac 3060

attggtcaga cacagattat cggtatgttc atacagatgc cgatcatgat cgcgtacgaa 3120

acagaccgtg ccaccggtga tggtataggg ctgcccatta aacacatgaa tacccgtgcc 3180

atgttcgaca atcacaattt catgaaaatc atgatgatgt tcaggaaaat ccgcctgcgg 3240

gagccggggt tctatcgcca cggacgcgtt accagacgga aaaaaatcca cactatgtaa 3300

tacggtcata ctggcctcct gatgtcgtca acacggcgaa atagtaatca cgaggtcagg 3360

ttcttacctt aaattttcga cggaaaacca cgtaaaaaac gtcgattttt caagatacag 3420

cgtgaatttt caggaaatgc ggtgagcatc acatcaccac aattcagcaa attgtgaaca 3480

tcatcacgtt catctttccc tggttgccaa tggcccattt tcctgtcagt aacgagaagg 3540

tcgcgaattc aggcgctttt tagactggtc gtaatgaaca attcttaaga aggagatata 3600

catatgaacc catatgcagt ttatgatgag attgaagaaa agaggttgga agatgagcac 3660

tacagggaaa ttatattaga gcaacaaggc atggatgccg aaattattta taacaagctt 3720

cctgagcttg ctggaatttt ttcaatcgaa actaacaaac tctttggcga attactcacg 3780

gagaacgatg aagccgcaga gctcgttaat tctcttttgt atgaactaag cttaatgaaa 3840

gtaaaaatgg aggatatatg aaggaggcca tcatatgtcc gcaagaaacg ttgcgtccgt 3900

atcgcacgat caaaatttgc atgtgcttcc gcatgcggcc acctcgacca gcgccttggt 3960

cctagacggc gacagcctgg acaagatgat gcgcctggcc gaggtcatgg cgaccgggcg 4020

agcaactctg ccgaagcact tcaacggcaa ctccgccgat tgcctggccg tcgtcatgca 4080

ggccatgcag tggaggatga acccgtttgc cgtcgcacag aaaacccatc tggtgaatgg 4140

cgtgctcggt tacgaggcgc agctggtcaa cgccgtgatt acaacctgcg cacctgtcgt 4200

ggatcgcttg cactacgagt ggtacggcaa ctgggaaaag gtcatcggca agtttgtgat 4260

caaaaacagc gacaagggtg agtaccggca gccaggctgg aagcttgccg acgaggaagg 4320

cctgggcgtg aaggtttggg cgacattccg cggagaagac gagcccaggg tgttggagct 4380

gctactggcc caggcgcgta cacgcaacag cacgctctgg gccgacgatc ctcgccagca 4440

gctcgcgtac ctggcaacca aacgctggtc ccgcctctac tgcccggacg tgattctggg 4500

ggtttacagc ccagatgagc tggaggaaag cacccctcgg taccgtgatg tttccccaca 4560

gccggagcag caagggtccg agcttcccgc ctacgaggac gaaaagttca aaacgatgtt 4620

tcccaaatgg caggagggaa tcgacaccgg aaagaccgac accgaaagcc tgatcgcctt 4680

cttggaatcg aaatacaccc tcagtgccga tcagatcgac cgaatcaacc agatggctcc 4740

tatcgccgga gaaaccgcat gaagacgcat aaagtcaccc agggcagtgc cgaatggcac 4800

gcgctccgcg ccaactatcg caccgcgtct gaagcaccgg ttatgatggg cgcatccaag 4860

cagatgaagc gcaccgagct gcttcatgcc aagaagaccg ggctcgaccg ggacatttcg 4920

tggtgggtcc agaagtactt gttcgacaag ggtcacgaag ccgaagcgct ggcgcgacca 4980

attctcgaag gtcgaatcgg cgaagacctg ttcccggttg tgggcaccga aggtgacctg 5040

ctggcctccc tcgacggctg cacaatgatg ggcgagatcg ttttcgagca caaaatgtgg 5100

aacgagcagc tcgccgccga cgtgcgtgcg ggcgatctgg atgcgcatta ctactggcag 5160

ctggagcagc aactgctggt gtcaggtgct gagaaagtga ttttcgtttg ctcggacggg 5220

accgaagaca acttcgtctc gatggagtac ttcccggtga acggtcgggc ggcgaaactg 5280

gtcgccggct ggaaacagtt cgaagtcgac cttgaagcgt atgagccaac tgaggccgtg 5340

gtggaggcag ttgggaagac tccggacgcg ctgccagcgc tgcgcatcga agtaaccgga 5400

atggtcactg cgagcaacct ggagcagttc aaggcccatt cgctggccgt gttttcggcg 5460

atcaacactg acctgcagac cgaccagcac ttcgctgatg cagagaaaac agtgaagtgg 5520

tgtggcgagg tcgaggagcg cctggaagca gccaagcaac atgcgctgag ccagacggaa 5580

accatcgacg ccctcttccg caccatcgac gaaatatcgg agcaggcccg tcgcaaacgg 5640

cttgagctgg acaaactggt gaaagctcgc aaggtggcca ttcgcgaaga gatcgttctg 5700

aaagcgaaag cagcgctgcg tgatcatctg gacaagatca acacctcgtt cggcggcaag 5760

gtgcggctgc cggagatccc cgcggacttc gccggcgcga tcaaaggcaa aaaaaatatc 5820

gccagtctgc gtgacgccgc cgactctgaa ctggcccggg ccaagatcga ggccagccag 5880

atcggcgacg gtatccgcgc caacttggaa agcttgcgct cgctcgccgt ggatcatgcg 5940

tttctgttca acgatgctca gcagatcgtg ctcaagaaca atgacgacct ggtggccctc 6000

atcaaggtgc ggatcaacga gcacaagcag gccgaggaag cgaaagagct ggcgcagcgt 6060

gaacgtattc gtgctgaaga atcagcgaag cttgcggctg ccgccgaagc ggaacaagtt 6120

gccgaagcgg aaaaggcaac agccaacgcg cccgcgccgc aagctgctga ggcatccaaa 6180

ccagtcgaac aaccagctcc gagaatgtcg gccgtcaccc catctgcgaa ggtgccgccc 6240

aagcctacaa agctcgaagc gaacgtcacc gaccttcatg cgctggtaaa agccgtctac 6300

gaaggtcgtg cgccgatatc ggtcctcaca gtgaactggg gcgcgctgga cgaccttgtc 6360

cacattcaag gcgctgactt ccaaatggac ggagtaacca tcacgcaggt ggcagcatga 6420

tgtacaagta agcttaccgg tttattgact accggaagca gtgtgaccgt gtgcttctca 6480

aatgcctgag gccagtttgc tcaggctctc cccgtggagg taataattga cgatatgatc 6540

atttattctg cctcccagag cctgataaaa acggtgaatc cgttagcgag gtgccgccgg 6600

cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga 6660

caaggtatag ggcggcgagg cggctacagc cgatagtctg gaacagcgca cttacgggtt 6720

gctgcgcaac ccaagtgcta ccggcgcggc agcgtgaccc gtgtcggcgg ctccaacggc 6780

tcgccatcgt ccagaaaaca cggctcatcg ggcatcggca ggcgctgctg cccgcgccgt 6840

tcccattcct ccgtttcggt caaggctggc aggtctggtt ccatgcccgg aatgccgggc 6900

tggctgggcg gctcctcgcc ggggccggtc ggtagttgct gctcgcccgg atacagggtc 6960

gggatgcggc gcaggtcgcc atgccccaac agcgattcgt cctggtcgtc gtgatcaacc 7020

accacggcgg cactgaacac cgacaggcgc aactggtcgc ggggctggcc ccacgccacg 7080

cggtcattga ccacgtaggc cgacacggtg ccggggccgt tgagcttcac gacggagatc 7140

cagcgctcgg ccaccaagtc cttgactgcg tattggaccg tccgcaaaga acgtccgatg 7200

agcttggaaa gtgtcttctg gctgaccacc acggcgttct ggtggcccat ctgcgccacg 7260

aggtgatgca gcagcattgc cgccgtgggt ttcctcgcaa taagcccggc ccacgcctca 7320

tgcgctttgc gttccgtttg cacccagtga ccgggcttgt tcttggcttg aatgccgatt 7380

tctctggact gcgtggccat gcttatctcc atgcggtagg gtgccgcacg gttgcggcac 7440

catgcgcaat cagctgcaac ttttcggcag cgcgacaaca attatgcgtt gcgtaaaagt 7500

ggcagtcaat tacagatttt ctttaaccta cgcaatgagc tattgcgggg ggtgccgcaa 7560

tgagctgttg cgtacccccc ttttttaagt tgttgatttt taagtctttc gcatttcgcc 7620

ctatatctag ttctttggtg cccaaagaag ggcacccctg cggggttccc ccacgccttc 7680

ggcgcggctc cccctccggc aaaaagtggc ccctccgggg cttgttgatc gactgcgcgg 7740

ccttcggcct tgcccaaggt ggcgctgccc ccttggaacc cccgcactcg ccgccgtgag 7800

gctcgggggg caggcgggcg ggcttcgcct tcgactgccc ccactcgcat aggcttgggt 7860

cgttccaggc gcgtcaaggc caagccgctg cgcggtcgct gcgcgagcct tgacccgcct 7920

tccacttggt gtccaaccgg caagcgaagc gcgcaggccg caggccggag gcttttcccc 7980

agagaaaatt aaaaaaattg atggggcaag gccgcaggcc gcgcagttgg agccggtggg 8040

tatgtggtcg aaggctgggt agccggtggg caatccctgt ggtcaagctc gtgggcaggc 8100

gcagcctgtc catcagcttg tccagcaggg ttgtccacgg gccgagcgaa gcgagccagc 8160

cggtggccgc tcgcggccat cgtccacata tccacgggct ggcaagggag cgcagcgacc 8220

gcgcagggcg aagcccggag agcaagcccg tagggcgccg cagccgccgt aggcggtcac 8280

gactttgcga agcaaagtct agtgagtata ctcaagcatt gagtggcccg ccggaggcac 8340

cgccttgcgc tgcccccgtc gagccggttg gacaccaaaa gggaggggca ggcatggcgg 8400

catacgcgat catgcgatgc aagaagctgg cgaaaatggg caacgtggcg gccagtctca 8460

agcacgccta ccgcgagcgc gagacgccca acgctgacgc cagcaggacg ccagagaacg 8520

agcactgggc ggccagcagc accgatgaag cgatgggccg actgcgcgag ttgctgccag 8580

agaagcggcg caaggacgct gtgttggcgg tcgagtacgt catgacggcc agcccggaat 8640

ggtggaagtc ggccagccaa gaacagcagg cggcgttctt cgagaaggcg cacaagtggc 8700

tggcggacaa gtacggggcg gatcgcatcg tgacggccag catccaccgt gacgaaacca 8760

gcccgcacat gaccgcgttc gtggtgccgc tgacgcagga cggcaggctg tcggccaagg 8820

agttcatcgg caacaaagcg cagatgaccc gcgaccagac cacgtttgcg gccgctgtgg 8880

ccgatctagg gctgcaacgg ggcatcgagg gcagcaaggc acgtcacacg cgcattcagg 8940

cgttctacga ggccctggag cggccaccag tgggccacgt caccatcagc ccgcaagcgg 9000

tcgagccacg cgcctatgca ccgcagggat tggccgaaaa gctgggaatc tcaaagcgcg 9060

ttgagacgcc ggaagccgtg gccgaccggc tgacaaaagc ggttcggcag gggtatgagc 9120

ctgccctaca ggccgccgca ggagcgcgtg agatgcgcaa gaaggccgat caagcccaag 9180

agacggcccg ag 9192

<210> 27

<211>9351

<212> DNA

<213> Artificial sequence

<221> pBBR1-Rha-redGam-TEpsy-kan

<222>（1）…（9351）

<400> 27

accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat cgggatatgc 60

aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg 120

aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca catttccccg 180

aaaagtgcca cctgggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag 240

cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt 300

tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa 360

gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc 420

aagatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 480

cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 540

aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 600

tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 660

gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 720

aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 780

tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 840

ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 900

ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 960

cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 1020

tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 1080

ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 1140

tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 1200

tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 1260

ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 1320

accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 1380

atcctccagc gcggggatct catgctggag ttcttcgccc acccccatgg gcaaatatta 1440

tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga 1500

tggcttccat gtcggcagaa tgcttaatga attacaacag tttttatgca ttaatctttc 1560

tgcgaattga gatgacgcca ctggctgggc gtcatcccgg tttcccgggt aaacaccacc 1620

gaaaaatagt tactatcttc aaagccacat tcggtcgaaa tatcactgat taacaggcgg 1680

ctatgctgga gaagatattg cgcatgacac actctgacct gtcgcagata ttgattgatg 1740

gtcattccag tctgctggcg aaattgctga cgcaaaacgc gctcactgca cgatgcctca 1800

tcacaaaatt tatccagcgc aaagggactt ttcaggctag ccgccagccg ggtaatcagc 1860

ttatccagca acgtttcgct ggatgttggc ggcaacgaat cactggtgta acgatggcga 1920

ttcagcaaca tcaccaactg cccgaacagc aactcagcca tttcgttagc aaacggcaca 1980

tgctgactac tttcatgctc aagctgaccg ataacctgcc gcgcctgcgc catccccatg 2040

ctacctaagc gccagtgtgg ttgccctgcg ctggcgttaa atcccggaat cgccccctgc 2100

cagtcaagat tcagcttcag acgctccggg caataaataa tattctgcaa aaccagatcg 2160

ttaacggaag cgtaggagtg tttatcgtca gcatgaatgt aaaagagatc gccacgggta 2220

atgcgataag ggcgatcgtt gagtacatgc aggccattac cgcgccagac aatcaccagc 2280

tcacaaaaat catgtgtatg ttcagcaaag acatcttgcg gataacggtc agccacagcg 2340

actgcctgct ggtcgctggc aaaaaaatca tctttgagaa gttttaactg atgcgccacc 2400

gtggctacct cggccagaga acgaagttga ttattcgcaa tatggcgtac aaatacgttg 2460

agaagattcg cgttattgca gaaagccatc ccgtccctgg cgaatatcac gcggtgacca 2520

gttaaactct cggcgaaaaa gcgtcgaaaa gtggttactg tcgctgaatc cacagcgata 2580

ggcgatgtca gtaacgctgg cctcgctgtg gcgtagcaga tgtcgggctt tcatcagtcg 2640

caggcggttc aggtatcgct gaggcgtcag tcccgtttgc tgcttaagct gccgatgtag 2700

cgtacgcagt gaaagagaaa attgatccgc cacggcatcc caattcacct catcggcaaa 2760

atggtcctcc agccaggcca gaagcaagtt gagacgtgat gcgctgtttt ccaggttctc 2820

ctgcaaactg cttttacgca gcaagagcag taattgcata aacaagatct cgcgactggc 2880

ggtcgagggt aaatcatttt ccccttcctg ctgttccatc tgtgcaacca gctgtcgcac 2940

ctgctgcaat acgctgtggt taacgcgcca gtgagacgga tactgcccat ccagctcttg 3000

tggcagcaac tgattcagcc cggcgagaaa ctgaaatcga tccggcgagc gatacagcac 3060

attggtcaga cacagattat cggtatgttc atacagatgc cgatcatgat cgcgtacgaa 3120

acagaccgtg ccaccggtga tggtataggg ctgcccatta aacacatgaa tacccgtgcc 3180

atgttcgaca atcacaattt catgaaaatc atgatgatgt tcaggaaaat ccgcctgcgg 3240

gagccggggt tctatcgcca cggacgcgtt accagacgga aaaaaatcca cactatgtaa 3300

tacggtcata ctggcctcct gatgtcgtca acacggcgaa atagtaatca cgaggtcagg 3360

ttcttacctt aaattttcga cggaaaacca cgtaaaaaac gtcgattttt caagatacag 3420

cgtgaatttt caggaaatgc ggtgagcatc acatcaccac aattcagcaa attgtgaaca 3480

tcatcacgtt catctttccc tggttgccaa tggcccattt tcctgtcagt aacgagaagg 3540

tcgcgaattc aggcgctttt tagactggtc gtaatgaaca attcttaaga aggagatata 3600

catatggata ttaatactga aactgagatc aagcaaaagc attcactaac cccctttcct 3660

gttttcctaa tcagcccggc atttcgcggg cgatattttc acagctattt caggagttca 3720

gccatgaacg cttattacat tcaggatcgt cttgaggctc agagctgggc gcgtcactac 3780

cagcagctcg cccgtgaaga gaaagaggca gaactggcag acgacatgga aaaaggcctg 3840

ccccagcacc tgtttgaatc gctatgcatc gatcatttgc aacgccacgg ggccagcaaa 3900

aaatccatta cccgtgcgtt tgatgacgat gttgagtttc aggagcgcat ggcagaacac 3960

atccggtaca tggttgaaac cattgctcac caccaggttg atattgattc agaggtataa 4020

aggaggccat catatgtccg caagaaacgt tgcgtccgta tcgcacgatc aaaatttgca 4080

tgtgcttccg catgcggcca cctcgaccag cgccttggtc ctagacggcg acagcctgga 4140

caagatgatg cgcctggccg aggtcatggc gaccgggcga gcaactctgc cgaagcactt 4200

caacggcaac tccgccgatt gcctggccgt cgtcatgcag gccatgcagt ggaggatgaa 4260

cccgtttgcc gtcgcacaga aaacccatct ggtgaatggc gtgctcggtt acgaggcgca 4320

gctggtcaac gccgtgatta caacctgcgc acctgtcgtg gatcgcttgc actacgagtg 4380

gtacggcaac tgggaaaagg tcatcggcaa gtttgtgatc aaaaacagcg acaagggtga 4440

gtaccggcag ccaggctgga agcttgccga cgaggaaggc ctgggcgtga aggtttgggc 4500

gacattccgc ggagaagacg agcccagggt gttggagctg ctactggccc aggcgcgtac 4560

acgcaacagc acgctctggg ccgacgatcc tcgccagcag ctcgcgtacc tggcaaccaa 4620

acgctggtcc cgcctctact gcccggacgt gattctgggg gtttacagcc cagatgagct 4680

ggaggaaagc acccctcggt accgtgatgt ttccccacag ccggagcagc aagggtccga 4740

gcttcccgcc tacgaggacg aaaagttcaa aacgatgttt cccaaatggc aggagggaat 4800

cgacaccgga aagaccgaca ccgaaagcct gatcgccttc ttggaatcga aatacaccct 4860

cagtgccgat cagatcgacc gaatcaacca gatggctcct atcgccggag aaaccgcatg 4920

aagacgcata aagtcaccca gggcagtgcc gaatggcacg cgctccgcgc caactatcgc 4980

accgcgtctg aagcaccggt tatgatgggc gcatccaagc agatgaagcg caccgagctg 5040

cttcatgcca agaagaccgg gctcgaccgg gacatttcgt ggtgggtcca gaagtacttg 5100

ttcgacaagg gtcacgaagc cgaagcgctg gcgcgaccaa ttctcgaagg tcgaatcggc 5160

gaagacctgt tcccggttgt gggcaccgaa ggtgacctgc tggcctccct cgacggctgc 5220

acaatgatgg gcgagatcgt tttcgagcac aaaatgtgga acgagcagct cgccgccgac 5280

gtgcgtgcgg gcgatctgga tgcgcattac tactggcagc tggagcagca actgctggtg 5340

tcaggtgctg agaaagtgat tttcgtttgc tcggacggga ccgaagacaa cttcgtctcg 5400

atggagtact tcccggtgaa cggtcgggcg gcgaaactgg tcgccggctg gaaacagttc 5460

gaagtcgacc ttgaagcgta tgagccaact gaggccgtgg tggaggcagt tgggaagact 5520

ccggacgcgc tgccagcgct gcgcatcgaa gtaaccggaa tggtcactgc gagcaacctg 5580

gagcagttca aggcccattc gctggccgtg ttttcggcga tcaacactga cctgcagacc 5640

gaccagcact tcgctgatgc agagaaaaca gtgaagtggt gtggcgaggt cgaggagcgc 5700

ctggaagcag ccaagcaaca tgcgctgagc cagacggaaa ccatcgacgc cctcttccgc 5760

accatcgacg aaatatcgga gcaggcccgt cgcaaacggc ttgagctgga caaactggtg 5820

aaagctcgca aggtggccat tcgcgaagag atcgttctga aagcgaaagc agcgctgcgt 5880

gatcatctgg acaagatcaa cacctcgttc ggcggcaagg tgcggctgcc ggagatcccc 5940

gcggacttcg ccggcgcgat caaaggcaaa aaaaatatcg ccagtctgcg tgacgccgcc 6000

gactctgaac tggcccgggc caagatcgag gccagccaga tcggcgacgg tatccgcgcc 6060

aacttggaaa gcttgcgctc gctcgccgtg gatcatgcgt ttctgttcaa cgatgctcag 6120

cagatcgtgc tcaagaacaa tgacgacctg gtggccctca tcaaggtgcg gatcaacgag 6180

cacaagcagg ccgaggaagc gaaagagctg gcgcagcgtg aacgtattcg tgctgaagaa 6240

tcagcgaagc ttgcggctgc cgccgaagcg gaacaagttg ccgaagcgga aaaggcaaca 6300

gccaacgcgc ccgcgccgca agctgctgag gcatccaaac cagtcgaaca accagctccg 6360

agaatgtcgg ccgtcacccc atctgcgaag gtgccgccca agcctacaaa gctcgaagcg 6420

aacgtcaccg accttcatgc gctggtaaaa gccgtctacg aaggtcgtgc gccgatatcg 6480

gtcctcacag tgaactgggg cgcgctggac gaccttgtcc acattcaagg cgctgacttc 6540

caaatggacg gagtaaccat cacgcaggtg gcagcatgat gtacaagtaa gcttaccggt 6600

ttattgacta ccggaagcag tgtgaccgtg tgcttctcaa atgcctgagg ccagtttgct 6660

caggctctcc ccgtggaggt aataattgac gatatgatca tttattctgc ctcccagagc 6720

ctgataaaaa cggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg 6780

cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgaggc 6840

ggctacagcc gatagtctgg aacagcgcac ttacgggttg ctgcgcaacc caagtgctac 6900

cggcgcggca gcgtgacccg tgtcggcggc tccaacggct cgccatcgtc cagaaaacac 6960

ggctcatcgg gcatcggcag gcgctgctgc ccgcgccgtt cccattcctc cgtttcggtc 7020

aaggctggca ggtctggttc catgcccgga atgccgggct ggctgggcgg ctcctcgccg 7080

gggccggtcg gtagttgctg ctcgcccgga tacagggtcg ggatgcggcg caggtcgcca 7140

tgccccaaca gcgattcgtc ctggtcgtcg tgatcaacca ccacggcggc actgaacacc 7200

gacaggcgca actggtcgcg gggctggccc cacgccacgc ggtcattgac cacgtaggcc 7260

gacacggtgc cggggccgtt gagcttcacg acggagatcc agcgctcggc caccaagtcc 7320

ttgactgcgt attggaccgt ccgcaaagaa cgtccgatga gcttggaaag tgtcttctgg 7380

ctgaccacca cggcgttctg gtggcccatc tgcgccacga ggtgatgcag cagcattgcc 7440

gccgtgggtt tcctcgcaat aagcccggcc cacgcctcat gcgctttgcg ttccgtttgc 7500

acccagtgac cgggcttgtt cttggcttga atgccgattt ctctggactg cgtggccatg 7560

cttatctcca tgcggtaggg tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact 7620

tttcggcagc gcgacaacaa ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc 7680

tttaacctac gcaatgagct attgcggggg gtgccgcaat gagctgttgc gtacccccct 7740

tttttaagtt gttgattttt aagtctttcg catttcgccc tatatctagt tctttggtgc 7800

ccaaagaagg gcacccctgc ggggttcccc cacgccttcg gcgcggctcc ccctccggca 7860

aaaagtggcc cctccggggc ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg 7920

gcgctgcccc cttggaaccc ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg 7980

gcttcgcctt cgactgcccc cactcgcata ggcttgggtc gttccaggcg cgtcaaggcc 8040

aagccgctgc gcggtcgctg cgcgagcctt gacccgcctt ccacttggtg tccaaccggc 8100

aagcgaagcg cgcaggccgc aggccggagg cttttcccca gagaaaatta aaaaaattga 8160

tggggcaagg ccgcaggccg cgcagttgga gccggtgggt atgtggtcga aggctgggta 8220

gccggtgggc aatccctgtg gtcaagctcg tgggcaggcg cagcctgtcc atcagcttgt 8280

ccagcagggt tgtccacggg ccgagcgaag cgagccagcc ggtggccgct cgcggccatc 8340

gtccacatat ccacgggctg gcaagggagc gcagcgaccg cgcagggcga agcccggaga 8400

gcaagcccgt agggcgccgc agccgccgta ggcggtcacg actttgcgaa gcaaagtcta 8460

gtgagtatac tcaagcattg agtggcccgc cggaggcacc gccttgcgct gcccccgtcg 8520

agccggttgg acaccaaaag ggaggggcag gcatggcggc atacgcgatc atgcgatgca 8580

agaagctggc gaaaatgggc aacgtggcgg ccagtctcaa gcacgcctac cgcgagcgcg 8640

agacgcccaa cgctgacgcc agcaggacgc cagagaacga gcactgggcg gccagcagca 8700

ccgatgaagc gatgggccga ctgcgcgagt tgctgccaga gaagcggcgc aaggacgctg 8760

tgttggcggt cgagtacgtc atgacggcca gcccggaatg gtggaagtcg gccagccaag 8820

aacagcaggc ggcgttcttc gagaaggcgc acaagtggct ggcggacaag tacggggcgg 8880

atcgcatcgt gacggccagc atccaccgtg acgaaaccag cccgcacatg accgcgttcg 8940

tggtgccgct gacgcaggac ggcaggctgt cggccaagga gttcatcggc aacaaagcgc 9000

agatgacccg cgaccagacc acgtttgcgg ccgctgtggc cgatctaggg ctgcaacggg 9060

gcatcgaggg cagcaaggca cgtcacacgc gcattcaggc gttctacgag gccctggagc 9120

ggccaccagt gggccacgtc accatcagcc cgcaagcggt cgagccacgc gcctatgcac 9180

cgcagggatt ggccgaaaag ctgggaatct caaagcgcgt tgagacgccg gaagccgtgg 9240

ccgaccggct gacaaaagcg gttcggcagg ggtatgagcc tgccctacag gccgccgcag 9300

gagcgcgtga gatgcgcaag aaggccgatc aagcccaaga gacggcccga g 9351

<210> 28

<211>8210

<212> DNA

<213> Artificial sequence

<221> pBBR1-Rha-BAS-kan

<222>（1）…（8210）

<400> 28

atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgggcaa cgtggcggcc 60

agtctcaagc acgcctaccg cgagcgcgag acgcccaacg ctgacgccag caggacgcca 120

gagaacgagc actgggcggc cagcagcacc gatgaagcga tgggccgact gcgcgagttg 180

ctgccagaga agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat gacggccagc 240

ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga gaaggcgcac 300

aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat ccaccgtgac 360

gaaaccagcc cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg caggctgtcg 420

gccaaggagt tcatcggcaa caaagcgcag atgacccgcg accagaccac gtttgcggcc 480

gctgtggccg atctagggct gcaacggggc atcgagggca gcaaggcacg tcacacgcgc 540

attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac catcagcccg 600

caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct gggaatctca 660

aagcgcgttg agacgccgga agccgtggcc gaccggctga caaaagcggt tcggcagggg 720

tatgagcctg ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa ggccgatcaa 780

gcccaagaga cggcccgaga ccttcgggag cgcctgaagc ccgttctgga cgccctgggg 840

ccgttgaatc gggatatgca ggccaaggcc gccgcgatca tcaaggccgt gggcgaaaag 900

ctgctgacgg aacagcggga agtccagcgc cagaaacagg cccagcgcca gcaggaacgc 960

gggcgcgcac atttccccga aaagtgccac ctgggatgaa tgtcagctac tgggctatct 1020

ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc 1080

gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc 1140

cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga 1200

tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga 1260

ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 1320

atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 1380

aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 1440

acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 1500

acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 1560

tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 1620

ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 1680

agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 1740

atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 1800

aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 1860

gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 1920

cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 1980

tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 2040

agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 2100

atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 2160

ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 2220

cccccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg 2280

ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt 2340

ttttatgcat taatctttct gcgaattgag atgacgccac tggctgggcg tcatcccggt 2400

ttcccgggta aacaccaccg aaaaatagtt actatcttca aagccacatt cggtcgaaat 2460

atcactgatt aacaggcggc tatgctggag aagatattgc gcatgacaca ctctgacctg 2520

tcgcagatat tgattgatgg tcattccagt ctgctggcga aattgctgac gcaaaacgcg 2580

ctcactgcac gatgcctcat cacaaaattt atccagcgca aagggacttt tcaggctagc 2640

cgccagccgg gtaatcagct tatccagcaa cgtttcgctg gatgttggcg gcaacgaatc 2700

actggtgtaa cgatggcgat tcagcaacat caccaactgc ccgaacagca actcagccat 2760

ttcgttagca aacggcacat gctgactact ttcatgctca agctgaccga taacctgccg 2820

cgcctgcgcc atccccatgc tacctaagcg ccagtgtggt tgccctgcgc tggcgttaaa 2880

tcccggaatc gccccctgcc agtcaagatt cagcttcaga cgctccgggc aataaataat 2940

attctgcaaa accagatcgt taacggaagc gtaggagtgt ttatcgtcag catgaatgta 3000

aaagagatcg ccacgggtaa tgcgataagg gcgatcgttg agtacatgca ggccattacc 3060

gcgccagaca atcaccagct cacaaaaatc atgtgtatgt tcagcaaaga catcttgcgg 3120

ataacggtca gccacagcga ctgcctgctg gtcgctggca aaaaaatcat ctttgagaag 3180

ttttaactga tgcgccaccg tggctacctc ggccagagaa cgaagttgat tattcgcaat 3240

atggcgtaca aatacgttga gaagattcgc gttattgcag aaagccatcc cgtccctggc 3300

gaatatcacg cggtgaccag ttaaactctc ggcgaaaaag cgtcgaaaag tggttactgt 3360

cgctgaatcc acagcgatag gcgatgtcag taacgctggc ctcgctgtgg cgtagcagat 3420

gtcgggcttt catcagtcgc aggcggttca ggtatcgctg aggcgtcagt cccgtttgct 3480

gcttaagctg ccgatgtagc gtacgcagtg aaagagaaaa ttgatccgcc acggcatccc 3540

aattcacctc atcggcaaaa tggtcctcca gccaggccag aagcaagttg agacgtgatg 3600

cgctgttttc caggttctcc tgcaaactgc ttttacgcag caagagcagt aattgcataa 3660

acaagatctc gcgactggcg gtcgagggta aatcattttc cccttcctgc tgttccatct 3720

gtgcaaccag ctgtcgcacc tgctgcaata cgctgtggtt aacgcgccag tgagacggat 3780

actgcccatc cagctcttgt ggcagcaact gattcagccc ggcgagaaac tgaaatcgat 3840

ccggcgagcg atacagcaca ttggtcagac acagattatc ggtatgttca tacagatgcc 3900

gatcatgatc gcgtacgaaa cagaccgtgc caccggtgat ggtatagggc tgcccattaa 3960

acacatgaat acccgtgcca tgttcgacaa tcacaatttc atgaaaatca tgatgatgtt 4020

caggaaaatc cgcctgcggg agccggggtt ctatcgccac ggacgcgtta ccagacggaa 4080

aaaaatccac actatgtaat acggtcatac tggcctcctg atgtcgtcaa cacggcgaaa 4140

tagtaatcac gaggtcaggt tcttacctta aattttcgac ggaaaaccac gtaaaaaacg 4200

tcgatttttc aagatacagc gtgaattttc aggaaatgcg gtgagcatca catcaccaca 4260

attcagcaaa ttgtgaacat catcacgttc atctttccct ggttgccaat ggcccatttt 4320

cctgtcagta acgagaaggt cgcgaattca ggcgcttttt agactggtcg taatgaacaa 4380

ttcttaagaa ggagatatac atatgggaac tgcactaaca ccgctcctga cgaagttcgc 4440

cacgcgctac gagatgggta ccacgcctga agaagtggcg aacacgctca agcagacctg 4500

tttcaagggc caggtcaatg attcgcagat ggtcgccctg ctgatcgtgg cagaccagta 4560

caaactgaac cccttcacca aggagttgta cgcattcccc gacaagaaca acggcatcgt 4620

gccagttgtt ggcgtggatg gctgggctcg gatcatcaac gagaacccac agttcgatgg 4680

catggaattc tcaatggacc agcagggaac cgaatgcacc tgcaagatct atcggaagga 4740

ccgcagccat gccatcagcg cgactgagta catggccgag tgcaagcgga acacccagcc 4800

ttggcagtcc catccgcgcc ggatgcttcg ccacaaggca atgatccagt gcgcacgcct 4860

cgcgttcggg ttcgccggca tatacgacca ggacgaggcc gagcggatcg ttgaacgaga 4920

cgtcactccc gcagaacagt acgaggacgt cagcgaggcg atctgcctca tcaaggactc 4980

cccaacaatg gaagacttgc agtcagcatt cagcaatgcc tggaaagcct acaagacaaa 5040

gggcgcgcgc gaccagctta cggcggccaa ggaccagcgg aagaaagaac tgctggacgc 5100

ccctatagac gttgaattcg aggagaccgg cgatgatcga gcagcgtagt gatgaatggt 5160

tcgcacagcg cctggggcgg gtgacggcca gcaaggtcaa ggatgtgatg gcaaaggggc 5220

gcagtggcgc cccttctgct acccgccaga actacatgat gcagctcctg tgcgagcgcc 5280

tgaccggcaa gcgcgaggaa ggattcacca gcgccgcaat gcagcgtggt accgacctgg 5340

agccgattgc ccgctcggca tacgagttca atgcaggcgt aatgacgatc gaaacaggcc 5400

tgatcatcca tccgcgaatc gatggatttg gcgcgtcgcc agatggcctc gcgggggagc 5460

atgggctcgt cgagattaaa tgcccgtcta ccgcaaccca catctacacg atgcagtcgg 5520

gcaagcacga cccacagtac gagtggcaga tgctcgccca aatgtcatgc agcggccgcg 5580

agtgggtcga cttcgtgagc ttcgacgacc gtctgcctga tgaattgcag tacgtgtgct 5640

tccgctacca ccgcgacgag gaacgcattc gcgagatgga gtccgaggtt aaagcgttcc 5700

tggaagagtt ggcagagctt gaacatcaga tgcgagaacg catgaggaaa gcagcatgag 5760

tgcacaggag gggatcctat gagaggcgtt aacaaagtca ttctggttgg caacgtcggt 5820

ggtgacccgg aaactcgcta tatgcccaac ggcaatgcgg tgaccaacat caccctcgcc 5880

accagcgaga gctggaagga caagcagacc ggccagcaac aggagcgcac cgaatggcac 5940

cgcgtggtgt tcttcgggaa gttggcggag atcgtcgggc agcacgtaaa gaagggtcag 6000

caactgtacg tcgagggttc actcagaact cgaaagtggc aggcgcagga cggtcaggac 6060

cgctacacaa cagagatcat cgtcgacatg cacgggcaga tgcagatgtt tggcggaaag 6120

cctggaaatg aacaggcagc tcagagcagg tcatctactc agcagcagag tgcgcctcag 6180

cagcgtagcg ctcaggatga attcgacgac gatattcctt tataaggatc cgtgcactgt 6240

acaagtaagc ttaccggttt attgactacc ggaagcagtg tgaccgtgtg cttctcaaat 6300

gcctgaggcc agtttgctca ggctctcccc gtggaggtaa taattgacga tatgatcatt 6360

tattctgcct cccagagcct gataaaaacg gtgaatccgt tagcgaggtg ccgccggctt 6420

ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa 6480

ggtatagggc ggcgaggcgg ctacagccga tagtctggaa cagcgcactt acgggttgct 6540

gcgcaaccca agtgctaccg gcgcggcagc gtgacccgtg tcggcggctc caacggctcg 6600

ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc gcgccgttcc 6660

cattcctccg tttcggtcaa ggctggcagg tctggttcca tgcccggaat gccgggctgg 6720

ctgggcggct cctcgccggg gccggtcggt agttgctgct cgcccggata cagggtcggg 6780

atgcggcgca ggtcgccatg ccccaacagc gattcgtcct ggtcgtcgtg atcaaccacc 6840

acggcggcac tgaacaccga caggcgcaac tggtcgcggg gctggcccca cgccacgcgg 6900

tcattgacca cgtaggccga cacggtgccg gggccgttga gcttcacgac ggagatccag 6960

cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc gcaaagaacg tccgatgagc 7020

ttggaaagtg tcttctggct gaccaccacg gcgttctggt ggcccatctg cgccacgagg 7080

tgatgcagca gcattgccgc cgtgggtttc ctcgcaataa gcccggccca cgcctcatgc 7140

gctttgcgtt ccgtttgcac ccagtgaccg ggcttgttct tggcttgaat gccgatttct 7200

ctggactgcg tggccatgct tatctccatg cggtagggtg ccgcacggtt gcggcaccat 7260

gcgcaatcag ctgcaacttt tcggcagcgc gacaacaatt atgcgttgcg taaaagtggc 7320

agtcaattac agattttctt taacctacgc aatgagctat tgcggggggt gccgcaatga 7380

gctgttgcgt accccccttt tttaagttgt tgatttttaa gtctttcgca tttcgcccta 7440

tatctagttc tttggtgccc aaagaagggc acccctgcgg ggttccccca cgccttcggc 7500

gcggctcccc ctccggcaaa aagtggcccc tccggggctt gttgatcgac tgcgcggcct 7560

tcggccttgc ccaaggtggc gctgccccct tggaaccccc gcactcgccg ccgtgaggct 7620

cggggggcag gcgggcgggc ttcgccttcg actgccccca ctcgcatagg cttgggtcgt 7680

tccaggcgcg tcaaggccaa gccgctgcgc ggtcgctgcg cgagccttga cccgccttcc 7740

acttggtgtc caaccggcaa gcgaagcgcg caggccgcag gccggaggct tttccccaga 7800

gaaaattaaa aaaattgatg gggcaaggcc gcaggccgcg cagttggagc cggtgggtat 7860

gtggtcgaag gctgggtagc cggtgggcaa tccctgtggt caagctcgtg ggcaggcgca 7920

gcctgtccat cagcttgtcc agcagggttg tccacgggcc gagcgaagcg agccagccgg 7980

tggccgctcg cggccatcgt ccacatatcc acgggctggc aagggagcgc agcgaccgcg 8040

cagggcgaag cccggagagc aagcccgtag ggcgccgcag ccgccgtagg cggtcacgac 8100

tttgcgaagc aaagtctagt gagtatactc aagcattgag tggcccgccg gaggcaccgc 8160

cttgcgctgc ccccgtcgag ccggttggac accaaaaggg aggggcaggc 8210

<210> 29

<211>8481

<212> DNA

<213> Artificial sequence

<221> pBBR1-Rha-pluGam-BAS-kan

<222>（1）…（8481）

<400> 29

atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgggcaa cgtggcggcc 60

agtctcaagc acgcctaccg cgagcgcgag acgcccaacg ctgacgccag caggacgcca 120

gagaacgagc actgggcggc cagcagcacc gatgaagcga tgggccgact gcgcgagttg 180

ctgccagaga agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat gacggccagc 240

ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga gaaggcgcac 300

aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat ccaccgtgac 360

gaaaccagcc cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg caggctgtcg 420

gccaaggagt tcatcggcaa caaagcgcag atgacccgcg accagaccac gtttgcggcc 480

gctgtggccg atctagggct gcaacggggc atcgagggca gcaaggcacg tcacacgcgc 540

attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac catcagcccg 600

caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct gggaatctca 660

aagcgcgttg agacgccgga agccgtggcc gaccggctga caaaagcggt tcggcagggg 720

tatgagcctg ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa ggccgatcaa 780

gcccaagaga cggcccgaga ccttcgggag cgcctgaagc ccgttctgga cgccctgggg 840

ccgttgaatc gggatatgca ggccaaggcc gccgcgatca tcaaggccgt gggcgaaaag 900

ctgctgacgg aacagcggga agtccagcgc cagaaacagg cccagcgcca gcaggaacgc 960

gggcgcgcac atttccccga aaagtgccac ctgggatgaa tgtcagctac tgggctatct 1020

ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc 1080

gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc 1140

cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga 1200

tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga 1260

ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 1320

atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 1380

aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 1440

acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 1500

acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 1560

tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 1620

ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 1680

agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 1740

atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 1800

aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 1860

gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 1920

cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 1980

tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 2040

agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 2100

atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 2160

ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 2220

cccccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg 2280

ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt 2340

ttttatgcat taatctttct gcgaattgag atgacgccac tggctgggcg tcatcccggt 2400

ttcccgggta aacaccaccg aaaaatagtt actatcttca aagccacatt cggtcgaaat 2460

atcactgatt aacaggcggc tatgctggag aagatattgc gcatgacaca ctctgacctg 2520

tcgcagatat tgattgatgg tcattccagt ctgctggcga aattgctgac gcaaaacgcg 2580

ctcactgcac gatgcctcat cacaaaattt atccagcgca aagggacttt tcaggctagc 2640

cgccagccgg gtaatcagct tatccagcaa cgtttcgctg gatgttggcg gcaacgaatc 2700

actggtgtaa cgatggcgat tcagcaacat caccaactgc ccgaacagca actcagccat 2760

ttcgttagca aacggcacat gctgactact ttcatgctca agctgaccga taacctgccg 2820

cgcctgcgcc atccccatgc tacctaagcg ccagtgtggt tgccctgcgc tggcgttaaa 2880

tcccggaatc gccccctgcc agtcaagatt cagcttcaga cgctccgggc aataaataat 2940

attctgcaaa accagatcgt taacggaagc gtaggagtgt ttatcgtcag catgaatgta 3000

aaagagatcg ccacgggtaa tgcgataagg gcgatcgttg agtacatgca ggccattacc 3060

gcgccagaca atcaccagct cacaaaaatc atgtgtatgt tcagcaaaga catcttgcgg 3120

ataacggtca gccacagcga ctgcctgctg gtcgctggca aaaaaatcat ctttgagaag 3180

ttttaactga tgcgccaccg tggctacctc ggccagagaa cgaagttgat tattcgcaat 3240

atggcgtaca aatacgttga gaagattcgc gttattgcag aaagccatcc cgtccctggc 3300

gaatatcacg cggtgaccag ttaaactctc ggcgaaaaag cgtcgaaaag tggttactgt 3360

cgctgaatcc acagcgatag gcgatgtcag taacgctggc ctcgctgtgg cgtagcagat 3420

gtcgggcttt catcagtcgc aggcggttca ggtatcgctg aggcgtcagt cccgtttgct 3480

gcttaagctg ccgatgtagc gtacgcagtg aaagagaaaa ttgatccgcc acggcatccc 3540

aattcacctc atcggcaaaa tggtcctcca gccaggccag aagcaagttg agacgtgatg 3600

cgctgttttc caggttctcc tgcaaactgc ttttacgcag caagagcagt aattgcataa 3660

acaagatctc gcgactggcg gtcgagggta aatcattttc cccttcctgc tgttccatct 3720

gtgcaaccag ctgtcgcacc tgctgcaata cgctgtggtt aacgcgccag tgagacggat 3780

actgcccatc cagctcttgt ggcagcaact gattcagccc ggcgagaaac tgaaatcgat 3840

ccggcgagcg atacagcaca ttggtcagac acagattatc ggtatgttca tacagatgcc 3900

gatcatgatc gcgtacgaaa cagaccgtgc caccggtgat ggtatagggc tgcccattaa 3960

acacatgaat acccgtgcca tgttcgacaa tcacaatttc atgaaaatca tgatgatgtt 4020

caggaaaatc cgcctgcggg agccggggtt ctatcgccac ggacgcgtta ccagacggaa 4080

aaaaatccac actatgtaat acggtcatac tggcctcctg atgtcgtcaa cacggcgaaa 4140

tagtaatcac gaggtcaggt tcttacctta aattttcgac ggaaaaccac gtaaaaaacg 4200

tcgatttttc aagatacagc gtgaattttc aggaaatgcg gtgagcatca catcaccaca 4260

attcagcaaa ttgtgaacat catcacgttc atctttccct ggttgccaat ggcccatttt 4320

cctgtcagta acgagaaggt cgcgaattca ggcgcttttt agactggtcg taatgaacaa 4380

ttcttaagaa ggagatatac atatgaaccc atatgcagtt tatgatgaga ttgaagaaaa 4440

gaggttggaa gatgagcact acagggaaat tatattagag caacaaggca tggatgccga 4500

aattatttat aacaagcttc ctgagcttgc tggaattttt tcaatcgaaa ctaacaaact 4560

ctttggcgaa ttactcacgg agaacgatga agccgcagag ctcgttaatt ctcttttgta 4620

tgaactaagc ttaatgaaag taaaaatgga ggatatatga aggaggacta gtgatgggaa 4680

ctgcactaac accgctcctg acgaagttcg ccacgcgcta cgagatgggt accacgcctg 4740

aagaagtggc gaacacgctc aagcagacct gtttcaaggg ccaggtcaat gattcgcaga 4800

tggtcgccct gctgatcgtg gcagaccagt acaaactgaa ccccttcacc aaggagttgt 4860

acgcattccc cgacaagaac aacggcatcg tgccagttgt tggcgtggat ggctgggctc 4920

ggatcatcaa cgagaaccca cagttcgatg gcatggaatt ctcaatggac cagcagggaa 4980

ccgaatgcac ctgcaagatc tatcggaagg accgcagcca tgccatcagc gcgactgagt 5040

acatggccga gtgcaagcgg aacacccagc cttggcagtc ccatccgcgc cggatgcttc 5100

gccacaaggc aatgatccag tgcgcacgcc tcgcgttcgg gttcgccggc atatacgacc 5160

aggacgaggc cgagcggatc gttgaacgag acgtcactcc cgcagaacag tacgaggacg 5220

tcagcgaggc gatctgcctc atcaaggact ccccaacaat ggaagacttg cagtcagcat 5280

tcagcaatgc ctggaaagcc tacaagacaa agggcgcgcg cgaccagctt acggcggcca 5340

aggaccagcg gaagaaagaa ctgctggacg cccctataga cgttgaattc gaggagaccg 5400

gcgatgatcg agcagcgtag tgatgaatgg ttcgcacagc gcctggggcg ggtgacggcc 5460

agcaaggtca aggatgtgat ggcaaagggg cgcagtggcg ccccttctgc tacccgccag 5520

aactacatga tgcagctcct gtgcgagcgc ctgaccggca agcgcgagga aggattcacc 5580

agcgccgcaa tgcagcgtgg taccgacctg gagccgattg cccgctcggc atacgagttc 5640

aatgcaggcg taatgacgat cgaaacaggc ctgatcatcc atccgcgaat cgatggattt 5700

ggcgcgtcgc cagatggcct cgcgggggag catgggctcg tcgagattaa atgcccgtct 5760

accgcaaccc acatctacac gatgcagtcg ggcaagcacg acccacagta cgagtggcag 5820

atgctcgccc aaatgtcatg cagcggccgc gagtgggtcg acttcgtgag cttcgacgac 5880

cgtctgcctg atgaattgca gtacgtgtgc ttccgctacc accgcgacga ggaacgcatt 5940

cgcgagatgg agtccgaggt taaagcgttc ctggaagagt tggcagagct tgaacatcag 6000

atgcgagaac gcatgaggaa agcagcatga gtgcacagga ggggatccta tgagaggcgt 6060

taacaaagtc attctggttg gcaacgtcgg tggtgacccg gaaactcgct atatgcccaa 6120

cggcaatgcg gtgaccaaca tcaccctcgc caccagcgag agctggaagg acaagcagac 6180

cggccagcaa caggagcgca ccgaatggca ccgcgtggtg ttcttcggga agttggcgga 6240

gatcgtcggg cagcacgtaa agaagggtca gcaactgtac gtcgagggtt cactcagaac 6300

tcgaaagtgg caggcgcagg acggtcagga ccgctacaca acagagatca tcgtcgacat 6360

gcacgggcag atgcagatgt ttggcggaaa gcctggaaat gaacaggcag ctcagagcag 6420

gtcatctact cagcagcaga gtgcgcctca gcagcgtagc gctcaggatg aattcgacga 6480

cgatattcct ttataaggat ccgtgcactg tacaagtaag cttaccggtt tattgactac 6540

cggaagcagt gtgaccgtgt gcttctcaaa tgcctgaggc cagtttgctc aggctctccc 6600

cgtggaggta ataattgacg atatgatcat ttattctgcc tcccagagcc tgataaaaac 6660

ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat 6720

gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgaggcg gctacagccg 6780

atagtctgga acagcgcact tacgggttgc tgcgcaaccc aagtgctacc ggcgcggcag 6840

cgtgacccgt gtcggcggct ccaacggctc gccatcgtcc agaaaacacg gctcatcggg 6900

catcggcagg cgctgctgcc cgcgccgttc ccattcctcc gtttcggtca aggctggcag 6960

gtctggttcc atgcccggaa tgccgggctg gctgggcggc tcctcgccgg ggccggtcgg 7020

tagttgctgc tcgcccggat acagggtcgg gatgcggcgc aggtcgccat gccccaacag 7080

cgattcgtcc tggtcgtcgt gatcaaccac cacggcggca ctgaacaccg acaggcgcaa 7140

ctggtcgcgg ggctggcccc acgccacgcg gtcattgacc acgtaggccg acacggtgcc 7200

ggggccgttg agcttcacga cggagatcca gcgctcggcc accaagtcct tgactgcgta 7260

ttggaccgtc cgcaaagaac gtccgatgag cttggaaagt gtcttctggc tgaccaccac 7320

ggcgttctgg tggcccatct gcgccacgag gtgatgcagc agcattgccg ccgtgggttt 7380

cctcgcaata agcccggccc acgcctcatg cgctttgcgt tccgtttgca cccagtgacc 7440

gggcttgttc ttggcttgaa tgccgatttc tctggactgc gtggccatgc ttatctccat 7500

gcggtagggt gccgcacggt tgcggcacca tgcgcaatca gctgcaactt ttcggcagcg 7560

cgacaacaat tatgcgttgc gtaaaagtgg cagtcaatta cagattttct ttaacctacg 7620

caatgagcta ttgcgggggg tgccgcaatg agctgttgcg tacccccctt ttttaagttg 7680

ttgattttta agtctttcgc atttcgccct atatctagtt ctttggtgcc caaagaaggg 7740

cacccctgcg gggttccccc acgccttcgg cgcggctccc cctccggcaa aaagtggccc 7800

ctccggggct tgttgatcga ctgcgcggcc ttcggccttg cccaaggtgg cgctgccccc 7860

ttggaacccc cgcactcgcc gccgtgaggc tcggggggca ggcgggcggg cttcgccttc 7920

gactgccccc actcgcatag gcttgggtcg ttccaggcgc gtcaaggcca agccgctgcg 7980

cggtcgctgc gcgagccttg acccgccttc cacttggtgt ccaaccggca agcgaagcgc 8040

gcaggccgca ggccggaggc ttttccccag agaaaattaa aaaaattgat ggggcaaggc 8100

cgcaggccgc gcagttggag ccggtgggta tgtggtcgaa ggctgggtag ccggtgggca 8160

atccctgtgg tcaagctcgt gggcaggcgc agcctgtcca tcagcttgtc cagcagggtt 8220

gtccacgggc cgagcgaagc gagccagccg gtggccgctc gcggccatcg tccacatatc 8280

cacgggctgg caagggagcg cagcgaccgc gcagggcgaa gcccggagag caagcccgta 8340

gggcgccgca gccgccgtag gcggtcacga ctttgcgaag caaagtctag tgagtatact 8400

caagcattga gtggcccgcc ggaggcaccg ccttgcgctg cccccgtcga gccggttgga 8460

caccaaaagg gaggggcagg c 8481

<210>30

<211>8640

<212> DNA

<213> Artificial sequence

<221> pBBR1-Rha-redGam-BAS-kan

<222>（1）…（8640）

<400> 30

atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgggcaa cgtggcggcc 60

agtctcaagc acgcctaccg cgagcgcgag acgcccaacg ctgacgccag caggacgcca 120

gagaacgagc actgggcggc cagcagcacc gatgaagcga tgggccgact gcgcgagttg 180

ctgccagaga agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat gacggccagc 240

ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga gaaggcgcac 300

aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat ccaccgtgac 360

gaaaccagcc cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg caggctgtcg 420

gccaaggagt tcatcggcaa caaagcgcag atgacccgcg accagaccac gtttgcggcc 480

gctgtggccg atctagggct gcaacggggc atcgagggca gcaaggcacg tcacacgcgc 540

attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac catcagcccg 600

caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct gggaatctca 660

aagcgcgttg agacgccgga agccgtggcc gaccggctga caaaagcggt tcggcagggg 720

tatgagcctg ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa ggccgatcaa 780

gcccaagaga cggcccgaga ccttcgggag cgcctgaagc ccgttctgga cgccctgggg 840

ccgttgaatc gggatatgca ggccaaggcc gccgcgatca tcaaggccgt gggcgaaaag 900

ctgctgacgg aacagcggga agtccagcgc cagaaacagg cccagcgcca gcaggaacgc 960

gggcgcgcac atttccccga aaagtgccac ctgggatgaa tgtcagctac tgggctatct 1020

ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc 1080

gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc 1140

cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga 1200

tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga 1260

ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 1320

atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 1380

aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 1440

acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 1500

acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 1560

tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 1620

ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 1680

agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 1740

atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 1800

aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 1860

gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 1920

cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 1980

tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 2040

agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 2100

atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 2160

ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 2220

cccccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg 2280

ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt 2340

ttttatgcat taatctttct gcgaattgag atgacgccac tggctgggcg tcatcccggt 2400

ttcccgggta aacaccaccg aaaaatagtt actatcttca aagccacatt cggtcgaaat 2460

atcactgatt aacaggcggc tatgctggag aagatattgc gcatgacaca ctctgacctg 2520

tcgcagatat tgattgatgg tcattccagt ctgctggcga aattgctgac gcaaaacgcg 2580

ctcactgcac gatgcctcat cacaaaattt atccagcgca aagggacttt tcaggctagc 2640

cgccagccgg gtaatcagct tatccagcaa cgtttcgctg gatgttggcg gcaacgaatc 2700

actggtgtaa cgatggcgat tcagcaacat caccaactgc ccgaacagca actcagccat 2760

ttcgttagca aacggcacat gctgactact ttcatgctca agctgaccga taacctgccg 2820

cgcctgcgcc atccccatgc tacctaagcg ccagtgtggt tgccctgcgc tggcgttaaa 2880

tcccggaatc gccccctgcc agtcaagatt cagcttcaga cgctccgggc aataaataat 2940

attctgcaaa accagatcgt taacggaagc gtaggagtgt ttatcgtcag catgaatgta 3000

aaagagatcg ccacgggtaa tgcgataagg gcgatcgttg agtacatgca ggccattacc 3060

gcgccagaca atcaccagct cacaaaaatc atgtgtatgt tcagcaaaga catcttgcgg 3120

ataacggtca gccacagcga ctgcctgctg gtcgctggca aaaaaatcat ctttgagaag 3180

ttttaactga tgcgccaccg tggctacctc ggccagagaa cgaagttgat tattcgcaat 3240

atggcgtaca aatacgttga gaagattcgc gttattgcag aaagccatcc cgtccctggc 3300

gaatatcacg cggtgaccag ttaaactctc ggcgaaaaag cgtcgaaaag tggttactgt 3360

cgctgaatcc acagcgatag gcgatgtcag taacgctggc ctcgctgtgg cgtagcagat 3420

gtcgggcttt catcagtcgc aggcggttca ggtatcgctg aggcgtcagt cccgtttgct 3480

gcttaagctg ccgatgtagc gtacgcagtg aaagagaaaa ttgatccgcc acggcatccc 3540

aattcacctc atcggcaaaa tggtcctcca gccaggccag aagcaagttg agacgtgatg 3600

cgctgttttc caggttctcc tgcaaactgc ttttacgcag caagagcagt aattgcataa 3660

acaagatctc gcgactggcg gtcgagggta aatcattttc cccttcctgc tgttccatct 3720

gtgcaaccag ctgtcgcacc tgctgcaata cgctgtggtt aacgcgccag tgagacggat 3780

actgcccatc cagctcttgt ggcagcaact gattcagccc ggcgagaaac tgaaatcgat 3840

ccggcgagcg atacagcaca ttggtcagac acagattatc ggtatgttca tacagatgcc 3900

gatcatgatc gcgtacgaaa cagaccgtgc caccggtgat ggtatagggc tgcccattaa 3960

acacatgaat acccgtgcca tgttcgacaa tcacaatttc atgaaaatca tgatgatgtt 4020

caggaaaatc cgcctgcggg agccggggtt ctatcgccac ggacgcgtta ccagacggaa 4080

aaaaatccac actatgtaat acggtcatac tggcctcctg atgtcgtcaa cacggcgaaa 4140

tagtaatcac gaggtcaggt tcttacctta aattttcgac ggaaaaccac gtaaaaaacg 4200

tcgatttttc aagatacagc gtgaattttc aggaaatgcg gtgagcatca catcaccaca 4260

attcagcaaa ttgtgaacat catcacgttc atctttccct ggttgccaat ggcccatttt 4320

cctgtcagta acgagaaggt cgcgaattca ggcgcttttt agactggtcg taatgaacaa 4380

ttcttaagaa ggagatatac atatggatat taatactgaa actgagatca agcaaaagca 4440

ttcactaacc ccctttcctg ttttcctaat cagcccggca tttcgcgggc gatattttca 4500

cagctatttc aggagttcag ccatgaacgc ttattacatt caggatcgtc ttgaggctca 4560

gagctgggcg cgtcactacc agcagctcgc ccgtgaagag aaagaggcag aactggcaga 4620

cgacatggaa aaaggcctgc cccagcacct gtttgaatcg ctatgcatcg atcatttgca 4680

acgccacggg gccagcaaaa aatccattac ccgtgcgttt gatgacgatg ttgagtttca 4740

ggagcgcatg gcagaacaca tccggtacat ggttgaaacc attgctcacc accaggttga 4800

tattgattca gaggtataaa ggaggactag tgatgggaac tgcactaaca ccgctcctga 4860

cgaagttcgc cacgcgctac gagatgggta ccacgcctga agaagtggcg aacacgctca 4920

agcagacctg tttcaagggc caggtcaatg attcgcagat ggtcgccctg ctgatcgtgg 4980

cagaccagta caaactgaac cccttcacca aggagttgta cgcattcccc gacaagaaca 5040

acggcatcgt gccagttgtt ggcgtggatg gctgggctcg gatcatcaac gagaacccac 5100

agttcgatgg catggaattc tcaatggacc agcagggaac cgaatgcacc tgcaagatct 5160

atcggaagga ccgcagccat gccatcagcg cgactgagta catggccgag tgcaagcgga 5220

acacccagcc ttggcagtcc catccgcgcc ggatgcttcg ccacaaggca atgatccagt 5280

gcgcacgcct cgcgttcggg ttcgccggca tatacgacca ggacgaggcc gagcggatcg 5340

ttgaacgaga cgtcactccc gcagaacagt acgaggacgt cagcgaggcg atctgcctca 5400

tcaaggactc cccaacaatg gaagacttgc agtcagcatt cagcaatgcc tggaaagcct 5460

acaagacaaa gggcgcgcgc gaccagctta cggcggccaa ggaccagcgg aagaaagaac 5520

tgctggacgc ccctatagac gttgaattcg aggagaccgg cgatgatcga gcagcgtagt 5580

gatgaatggt tcgcacagcg cctggggcgg gtgacggcca gcaaggtcaa ggatgtgatg 5640

gcaaaggggc gcagtggcgc cccttctgct acccgccaga actacatgat gcagctcctg 5700

tgcgagcgcc tgaccggcaa gcgcgaggaa ggattcacca gcgccgcaat gcagcgtggt 5760

accgacctgg agccgattgc ccgctcggca tacgagttca atgcaggcgt aatgacgatc 5820

gaaacaggcc tgatcatcca tccgcgaatc gatggatttg gcgcgtcgcc agatggcctc 5880

gcgggggagc atgggctcgt cgagattaaa tgcccgtcta ccgcaaccca catctacacg 5940

atgcagtcgg gcaagcacga cccacagtac gagtggcaga tgctcgccca aatgtcatgc 6000

agcggccgcg agtgggtcga cttcgtgagc ttcgacgacc gtctgcctga tgaattgcag 6060

tacgtgtgct tccgctacca ccgcgacgag gaacgcattc gcgagatgga gtccgaggtt 6120

aaagcgttcc tggaagagtt ggcagagctt gaacatcaga tgcgagaacg catgaggaaa 6180

gcagcatgag tgcacaggag gggatcctat gagaggcgtt aacaaagtca ttctggttgg 6240

caacgtcggt ggtgacccgg aaactcgcta tatgcccaac ggcaatgcgg tgaccaacat 6300

caccctcgcc accagcgaga gctggaagga caagcagacc ggccagcaac aggagcgcac 6360

cgaatggcac cgcgtggtgt tcttcgggaa gttggcggag atcgtcgggc agcacgtaaa 6420

gaagggtcag caactgtacg tcgagggttc actcagaact cgaaagtggc aggcgcagga 6480

cggtcaggac cgctacacaa cagagatcat cgtcgacatg cacgggcaga tgcagatgtt 6540

tggcggaaag cctggaaatg aacaggcagc tcagagcagg tcatctactc agcagcagag 6600

tgcgcctcag cagcgtagcg ctcaggatga attcgacgac gatattcctt tataaggatc 6660

cgtgcactgt acaagtaagc ttaccggttt attgactacc ggaagcagtg tgaccgtgtg 6720

cttctcaaat gcctgaggcc agtttgctca ggctctcccc gtggaggtaa taattgacga 6780

tatgatcatt tattctgcct cccagagcct gataaaaacg gtgaatccgt tagcgaggtg 6840

ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg 6900

aggcagacaa ggtatagggc ggcgaggcgg ctacagccga tagtctggaa cagcgcactt 6960

acgggttgct gcgcaaccca agtgctaccg gcgcggcagc gtgacccgtg tcggcggctc 7020

caacggctcg ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc 7080

gcgccgttcc cattcctccg tttcggtcaa ggctggcagg tctggttcca tgcccggaat 7140

gccgggctgg ctgggcggct cctcgccggg gccggtcggt agttgctgct cgcccggata 7200

cagggtcggg atgcggcgca ggtcgccatg ccccaacagc gattcgtcct ggtcgtcgtg 7260

atcaaccacc acggcggcac tgaacaccga caggcgcaac tggtcgcggg gctggcccca 7320

cgccacgcgg tcattgacca cgtaggccga cacggtgccg gggccgttga gcttcacgac 7380

ggagatccag cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc gcaaagaacg 7440

tccgatgagc ttggaaagtg tcttctggct gaccaccacg gcgttctggt ggcccatctg 7500

cgccacgagg tgatgcagca gcattgccgc cgtgggtttc ctcgcaataa gcccggccca 7560

cgcctcatgc gctttgcgtt ccgtttgcac ccagtgaccg ggcttgttct tggcttgaat 7620

gccgatttct ctggactgcg tggccatgct tatctccatg cggtagggtg ccgcacggtt 7680

gcggcaccat gcgcaatcag ctgcaacttt tcggcagcgc gacaacaatt atgcgttgcg 7740

taaaagtggc agtcaattac agattttctt taacctacgc aatgagctat tgcggggggt 7800

gccgcaatga gctgttgcgt accccccttt tttaagttgt tgatttttaa gtctttcgca 7860

tttcgcccta tatctagttc tttggtgccc aaagaagggc acccctgcgg ggttccccca 7920

cgccttcggc gcggctcccc ctccggcaaa aagtggcccc tccggggctt gttgatcgac 7980

tgcgcggcct tcggccttgc ccaaggtggc gctgccccct tggaaccccc gcactcgccg 8040

ccgtgaggct cggggggcag gcgggcgggc ttcgccttcg actgccccca ctcgcatagg 8100

cttgggtcgt tccaggcgcg tcaaggccaa gccgctgcgc ggtcgctgcg cgagccttga 8160

cccgccttcc acttggtgtc caaccggcaa gcgaagcgcg caggccgcag gccggaggct 8220

tttccccaga gaaaattaaa aaaattgatg gggcaaggcc gcaggccgcg cagttggagc 8280

cggtgggtat gtggtcgaag gctgggtagc cggtgggcaa tccctgtggt caagctcgtg 8340

ggcaggcgca gcctgtccat cagcttgtcc agcagggttg tccacgggcc gagcgaagcg 8400

agccagccgg tggccgctcg cggccatcgt ccacatatcc acgggctggc aagggagcgc 8460

agcgaccgcg cagggcgaag cccggagagc aagcccgtag ggcgccgcag ccgccgtagg 8520

cggtcacgac tttgcgaagc aaagtctagt gagtatactc aagcattgag tggcccgccg 8580

gaggcaccgc cttgcgctgc ccccgtcgag ccggttggac accaaaaggg aggggcaggc 8640

Claims (2)

1. A novel recombination system in pseudomonas is pseudomonas aeruginosa, pseudomonas syringae, pseudomonas fluorescens or/and pseudomonas putida, and the recombination system consists of a series of homologous recombination system expression plasmids which are recombination systems comprising a pBBR1 replication origin, a kanamycin resistance gene and a rhamnose inducible promoter;

The method is characterized in that:

The expression plasmid of the series of homologous recombination systems in the recombination system is as follows: pBBR1-Rha-BAS-kan, pBBR1-Rha-pluGam-BAS-kan, or pBBR 1-Rha-redGam-BAS-kan; wherein the nucleotide sequence of pBBR1-Rha-BAS-kan is shown in SEQ ID No. 28; the nucleotide sequence of pBBR1-Rha-pluGam-BAS-kan is shown in SEQ ID No. 29; the nucleotide sequence of pBBR1-Rha-redGam-BAS-kan is shown in SEQ ID No. 30; the B is a single-chain DNA annealing protein or a recombinase, the A is a 5 '-3' double-chain DNA exonuclease, the S is a single-chain DNA binding protein (SSB) derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa) phage vB _ PaeP _ Tr60_ Ab31, the Red gamma is an inhibitor protein derived from escherichia coli exonuclease recBCD of lambda phage, and the Plu gamma is an inhibitor protein derived from exonuclease recBCD of Photorhabdus luminescens prophage.

2. Use of the novel recombination system in Pseudomonas according to claim 1 for mediating homologous recombination between a short homology arm and a long homology arm in a different species of Pseudomonas for genetic modification of genomic DNA or plasmids, wherein the short homology arm is from 50bp to 150bp and the long homology arm is greater than 150 bp;

The method is characterized in that:

When the genome DNA or plasmid is genetically modified by mediating the homologous recombination between the short homology arm and the long homology arm, pBBR1-Rha-redGam-BAS-kan is selected from Pseudomonas aeruginosa (Pseudomonas aeruginosa), pBBR1-Rha-redGam-BAS-kan is selected from Pseudomonas syringae, pBBR1-Rha-BAS-kan is selected from Pseudomonas putida, and pBBR1-Rha-BAS-kan is selected from Pseudomonas fluorescens (Pseudomonas fluorescens).