CN106929528A - New recombination system and its application in pseudomonad - Google Patents

Abstract

The invention discloses new recombination system in a kind of pseudomonad, the recombination system is according to from operator (Orf38 pseudomonas aeruginosa (Pseudomonas aeruginosa) bacteriophage Ab31 similar with λ Red, Orf37 and Orf36), and from pseudomonas syringae B728a prophages (the Pseudomonas syringae pv.syringae B728a) operator (recTE similar with RecET_Psy) build.Recombination system is in addition to host specificity exonuclease and single stranded DNA annealing two genes of albumen, 3rd gene is that exonuclease recBCD suppresses albumen, and the 3rd gene or the 4th gene can also be derived from bacterial chromosome, bacterial plasmid and other single-stranded DNA binding protein SSB for originating.Function detection result shows that the heterological system that the similar operators of λ Red and the similar operators of RecET are constituted with Red γ/Plu γ can work in pseudomonad, and SSB albumen can significantly improve recombination efficiency.Efficient succinct plasmid modification and point mutation, the genomic modification of insert and delete can be carried out to pseudomonad using these recombination systems, has been had a extensive future.

Description

New recombination system and its application in pseudomonad

Technical field

The present invention relates to the recombination system in a kind of gram-Negative bacillus and its structure and application, especially a kind of false unit cell Recombination system and its structure in bacterium (pseudomonas aeruginosa, pseudomonas syringae, Pseudomonas fluorescens or/and pseudomonas putida) Build with application, belong to microbiological genetic engineering field.

Background technology

The recombination system of phage encoded can have very when modifying Prokaryotic genome and building eucaryon genetically modified organism It is widely applied.It needs only to 35 nucleotides as homology arm, using it is main include building mutant-point mutation, deletion, Insertion and replacement.Shorter homology arm is the main advantage of this technology, and this technology is named as recombined engineering.

In Escherichia coli, most widely used recombination system is λ Red systems and racRecET systems.λ Red systems profit It is homologous heavy to mediate wire and ring-shaped DNA molecule to carry out with the Red operators (Red α, Red β and Red γ) for coming from bacteriophage lambda Group.RacRecET systems mediate two linear DNA molecules using RecE the and RecT albumen of Rac prophges in Escherichia coli Generation homologous recombination.Red α and RecE are 5 ' -3 ' double-stranded DNA exonucleases, and Red β and RecT are single-stranded annealing albumen.Document Report Red α/Red β and RecE/RecT promotes the homologous recombination of double-stranded DNA by protein-protein interaction.

Red γ can suppress the 5 prime excision enzyme activity of RecBCD, and RecBCD is a multifunctional enzyme compound, and it can process place The DNA ends that reason double-strand break is produced.Red γ can form a DNA analog for dimer, with RecBCD system centers Sour excision enzyme and unwindase mutually and suppress the activity of these enzymes.Document report is in RecE/RecT or Red α/Red beta systems Middle addition Red γ can improve DNA recombination efficiencies.Plu γ from luminous bacillus (Photorhabdus luminescens) The shape and construction of duplex DNA can also be imitated, suppresses host's nucleic acid in vivo excision enzyme complex Plu0632/Plu0630/ Plu0633, so as to can effectively improve the efficiency of homologous recombination in vivo.

The structure of similar λ Red systems and racRecET systems, bacteriophage or prophage genome in other bacteriums Middle search simultaneously clones the operator containing exonuclease and single-stranded annealing albumen, and insertion can improve weight in this operator Red γ or Plu the γ GFPs of group efficiency, are built into a complete DNA recombination system, have been built in Escherichia coli This set recombination system is carried out genetic modification by the host that electroporated or engagement transfer imports operator source into rear.Perhaps The recombination system that many document reports are built by using bacteriophage recombination function operator, such as luminous bacillus, Xenorhabdus (Xenorhabdus), bacillus subtilis (B.subtilis), clostridium (Clostridium), pseudomonas syringae (P.syringae), mycobacterium tuberculosis (M.tuberculosis), Bacillus acidi lactici (Lactobacillus plantarum) etc. Deng, but host specificity causes the range of application of these recombination systems very narrow.

Pseudomonad is a kind of Gram-negative bacteria, aerobic condition growth, belongs to pseudomonadaceae, most study it is several Pseudomonad is human condition pathogenic bacteria pseudomonas aeruginosa (Pseudomonas aeruginosa), plant conditions pathogenic bacteria fourth Fragrant pseudomonad (Pseudomonassyringae), plant growth-promoting bacteria Pseudomonas fluorescens (Pseudomonas Fluorescens), the pseudomonas putida (Pseudomonas being also commonly used during biological prosthetic and biological control putida).Traditional gene manipulation techniques is real in vitro by technologies such as restriction enzyme, DNA ligase and PCR amplifications The modifications such as the now insertion of genes of interest, knockout and point mutation, but it is cumbersome and less efficient, due to restriction enzyme site Limitation and the research and transformation to pseudomonad genome are limited the shortcomings of cumbersome and inefficiency.Relevant vacation unit cell Recombination system and its structure in bacterium (pseudomonas aeruginosa, pseudomonas syringae, Pseudomonas fluorescens and pseudomonas putida) Research and patent with application are rarely reported.

The content of the invention

In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of pseudomonad, (pseudomonas aeruginosa, cloves are false Monad, Pseudomonas fluorescens or/and pseudomonas putida) in recombination system and its build with application.

In order to realize above-mentioned purpose of the invention, the invention provides following technical scheme：

New recombination system is made up of a series of homologous recombination system expression plasmids in pseudomonad of the present invention, its It is characterised by：The expression plasmid expresses an operator under inducible promoters, and the operator is removed comprising host specificity Outside exonuclease and single stranded DNA annealing two genes of albumen, the 3rd gene is derived from Escherichia coli lambda phagocytosis The Red γ of body, or from the Plu γ of luminous bacillus (Photorhabdus luminescens) prophge (Plu2934), or other external sources exonuclease recBCD suppress albumen；Or, the operator is removed comprising host specific Property exonuclease, single stranded DNA annealing albumen and external source or endogenous nucleic acid excision enzyme recBCD suppress outside three genes of albumen, 4th gene is derived from the single-stranded DNA binding protein SSB of bacterial chromosome, or the SSB on bacterial plasmid, or The SSB of bacteriophage is derived from, or from the SSB of bacterium prophge.

In above-mentioned pseudomonad in new recombination system, further the technical scheme of optimization is：In the recombination system Serial homology recombination system expression plasmid includes that pBBR1 replication orgins, kalamycin resistance gene and rhamnose induction type start The recombination system of son；Its name is respectively pBBR1-Rha-red_gba-Kan, and nucleotide sequence is as shown in SEQ ID No.23； PBBR1-Rha-plu_gba-kan, nucleotide sequence is as shown in SEQID No.24；PBBR1-Rha-TEpsy-kan, nucleotides Sequence is as shown in SEQ ID No.25；PBBR1-Rha-pluGam-TEpsy-kan, nucleotide sequence such as SEQ ID No.26 institutes Show；PBBR1-Rha-redGam-TEpsy-kan, nucleotide sequence is as shown in SEQ ID No.27；PBBR1-Rha-BAS-kan, Nucleotide sequence is as shown in SEQ ID No.28；PBBR1-Rha-pluGam-BAS-kan, nucleotide sequence such as SEQ ID Shown in No.29；PBBR1-Rha-redGam-BAS-kan, nucleotide sequence is as shown in SEQ ID No.30；Wherein, RecTEpsy The RecTEpsy in the prophge of pseudomonas syringae (Pseudomonas syringae) is derived from, B is that single stranded DNA is moved back Fiery albumen or be recombinase, A is 5 ' -3 ' double-stranded DNA exonucleases, and S is derived from pseudomonas aeruginosa The single-stranded DNA binding protein (SSB) of (Pseudomonas aeruginosa) bacteriophage vB_PaeP_Tr60_Ab31, Red γ are From the suppression albumen of the Escherichia coli nucleic acid excision enzyme recBCD of lambda bacteriophages, Plu γ are derived from luminous bacillus The suppression albumen of the exonuclease recBCD of (Photorhabdus luminescens) prophge.

In above-mentioned pseudomonad in new recombination system, the technical scheme of optimization is：Series in the recombination system Homologous recombination system expression plasmid is：PBBR1-Rha-BAS-kan, pBBR1-Rha-pluGam-TEpsy-kan, pBBR1- Rha-redGam-TEpsy-kan, pBBR1-Rha-redGam-BAS-kan or pBBR1-Rha-pluGam-BAS-kan.

In above-mentioned pseudomonad in new recombination system, it is false single that the pseudomonad is preferably pseudomonas aeruginosa, cloves Born of the same parents bacterium, Pseudomonas fluorescens or/and pseudomonas putida.

New recombination system leads short homology arm and length in pseudomonas intermediary not of the same race in pseudomonad of the present invention Homologous recombination between homology arm carries out the application in genomic DNA or plasmid genetic modification.Wherein, the short homology arm refers to The homology arm of 50bp-150bp, the homology arm long refers to the homology arm more than 150bp.

Optimal recombination system is that different, optimal restructuring conditions is also differed in various bacterial strains, carries out condition optimizing Such as competent cell Optimization of preparation, the optimization of DNA inversion quantities, antibiotic selection concentration optimization, homology arm length optimization etc. Recombination efficiency can be improved, efficient genetic modification is carried out further with the optimal recombination system of different strains.

Homologous recombination between the above-mentioned short homology arm of mediation and homology arm long carries out genomic DNA or plasmid genetic modification When, the preferred pBBR1-Rha-redGam-BAS-kan plasmids in pseudomonas aeruginosa (Pseudomonas aeruginosa), Preferred pBBR1-Rha-redGam-BAS-kan plasmids, pBBR1- in pseudomonas syringae (Pseudomonassyringae) Rha-redGam-TEpsy-kan plasmids or pBBR1-Rha-pluGam-TEpsy-kan plasmids, in pseudomonas putida Preferred pBBR1-Rha-BAS-kan plasmids in (Pseudomonas putida), in Pseudomonas fluorescens (Pseudomonas Fluorescens) preferred pBBR1-Rha-BAS-kan plasmids or pBBR1-Rha-pluGam-TEpsy-kan plasmids.

The present invention reports a series of recombination systems that can apply to pseudomonad, especially pseudomonas aeruginosa, Optimal recombination system is built in pseudomonas syringae, Pseudomonas fluorescens and pseudomonas putida, these recombination systems come There is recombination function from two host specificities in Pseudomonas aeruginosa phage Ab31 and pseudomonas syringae prophage Operator.BLAST analysis displays Orf38, Orf37 and Orf36 are respectively Red β, Red α and single-stranded DNA binding protein (SSB) Homologous protein.Reconstruction experiment result shows that the heterological system with Red γ/Plu γ can be in some pseudomonads very well Work, while find SSB albumen can significantly improve recombination efficiency.Using these recombination systems, we are successfully to false single Born of the same parents bacterium has carried out efficient succinct plasmid modification and point mutation, the genomic modification of insert and delete, has a extensive future.

Brief description of the drawings

Fig. 1：The recombination efficiency of different recombinase combinations compares in pseudomonad.

(A) schematic diagram that wire loop is recombinated in pseudomonad.PCR primer is homology arm (blueness) of the two ends with 100bp, in Between be gentamicin resistance gene (Genta), replace and the gene of correlation is recombinated in expression plasmid form new plasmid.

(B) pseudomonas aeruginosa expresses pBBR1-Rha-red_gba-Kan, pBBR1-Rha-plu_ under the conditions of 30 DEG C 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 Carry out wire loop recombination efficiency result of the comparison.

(C) a series of pseudomonas aeruginosa recombination systems in expression (B) under the conditions of 37 DEG C carry out wire loop recombination efficiency ratio Compared with result.

(D) a series of Pseudomonas fluorescens recombination systems in expression (B) under the conditions of 30 DEG C carry out wire loop recombination efficiency ratio Compared with result.

(E) a series of pseudomonas syringae recombination systems in expression (B) under the conditions of 30 DEG C carry out wire loop recombination efficiency ratio Compared with result.

(F) pseudomonas putida expresses pBBR1-Rha-red_gba-Kan, pBBR1-Rha-plu_ under the conditions of 30 DEG C 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 Carry out wire loop recombination efficiency result of the comparison.

Fig. 2：Pseudomonad electricity turns the optimization of condition.

(A), (B), (C) and (D) is pseudomonas aeruginosa, pseudomonas putida, pseudomonas syringae and Pseudomonas The growth curve of bacterium.Initial bacterial concentration OD600 is 0.085.

(E), (F), (G) and (H) is respectively false pseudomonas aeruginosa, pseudomonas putida, pseudomonas syringae and fluorescence The transformation efficiency of monad different time.

Fig. 3：The optimization of conversion amount of DNA and homologous arm lengths when being recombinated in pseudomonad.

Pseudomonas aeruginosa carries pBBR1-Rha-pluGam-TEpsy-kan (A), and pseudomonas putida carries pBBR1- Rha-BAS-kan systems (B), pseudomonas syringae carries pBBR1-Rha-BAS-kan (C), and Pseudomonas fluorescens carries Influences of the conversion various concentrations DNA to recombination efficiency in pBBR1-Rha-redGam-TEpsy-kan (D).

Pseudomonas aeruginosa carries pBBR1-Rha-pluGam-TEpsy-kan (E), and pseudomonas putida carries pBBR1- Rha-BAS-kan (F), pseudomonas syringae carries pBBR1-Rha-BAS-kan (G), and Pseudomonas fluorescens carries pBBR1- Influence of the PCR primer of conversion different length homology arm to recombination efficiency in Rha-redGam-TEpsy-kan (H).

Fig. 4：Bacterium colony PCR identifications after carrying out genomic modification by homologous recombination in pseudomonas aeruginosa.

(A) in pseudomonas aeruginosa the schematic diagram of genomic modification and bacterium colony PCR schematic diagram.

(B) phnAB genes are knocked out by inserting gentamicin selection markers, clone 1 and 2 is correct.

(C) recombinated using the locus specificity of Cre enzymes after phnAB genes are knocked out and delete gentamicin resistance screening mark Note, six clones are correct.

(D) lasB genes are knocked out by inserting gentamicin selection markers, clone 1 and 2 is correct.

(E) recombinated using the locus specificity of Cre enzymes after lasB genes are knocked out and delete gentamicin resistance selection markers, Clone is correct.

(F) mdoH genes are knocked out by inserting gentamicin selection markers, clone is correct.

(G) recombinated using the locus specificity of Cre enzymes after mdoH genes are knocked out and delete gentamicin resistance selection markers, Four clones are correct.

Fig. 5：In pseudomonas aeruginosa point mutation is carried out using new recombination system.

(A) schematic diagram of schematic diagram and bacterium colony the PCR identification of point mutation is carried out in pseudomonas aeruginosa.

(B) SacB- gentamicin components are inserted in toxA target sites, swimming lane C is wild-type strain as negative control, swimming Road M is TakaraDL5000, and swimming lane 1 and 2 is recon, and clone 1 and 2 is correct.

(C) insertion point mutation is matched by single-stranded nucleotide, three clones are correct.

(D) sequence verification of mutant.

Fig. 6：The functional verification of SacB, in LB (left plate) and LB (right plate) containing 10% sucrose.

1. the wild-type strain of pseudomonas aeruginosa；Different mutation after 2-8.PAO1 insertion SacB- gentamicin components Clone.

Fig. 7：Bacterium colony PCR checkings after carrying out genetic modification in pseudomonas syringae and Pseudomonas fluorescens.PCR primer Across complete Insert Fragment.

(A) promoter (P is inserted before the biosynthesis gene of pseudomonas syringae prediction_genta)。

(B) the retS genes to Pseudomonas fluorescens pf5 are knocked out, and swimming lane 1 is insertion gentamicin-SacB components (Pf5- Δ retS-genta-loxM), swimming lane 2,3 is that the final mutant strain after locus specificity restructuring is carried out using Cre enzymes (Pf5-ΔretS)。

Fig. 8：Influences of the SSB to pBBR1-Rha-BA-kan and pBBR1-Rha-redGam-BA-kan System reorganization functions.

At Escherichia coli (A), SSB is studied in pseudomonas aeruginosa (B) and in pseudomonas syringae (C) to pBBR1- The influence of Rha-BA-kan and pBBR1-Rha-redGam-BA-kan recombination functions.

Specific embodiment

The present invention is described in further detail with reference to the accompanying drawings and examples, method is unless otherwise specified in embodiment Use conventional method, the reagent for using unless otherwise specified use conventional commercial reagent or the examination for configuring according to a conventional method Agent.

Reagent and instrument：

Reagent is mainly molecular biology experiment reagent in the present embodiment, and single-stranded nucleotide is to give birth to work biotechnology from Shanghai Co., Ltd synthesizes, and restriction enzyme, archaeal dna polymerase and DNA marker come from biotech firm of New England (New England Biolabs), antibiotic is bought from hero company (Invitrogen).

The preparation of the electric transformed cells for being recombinated：

Various expression plasmids will respectively be transformed into Escherichia coli GB05 and be purchased from German GeneBridges companies, the false list of verdigris Born of the same parents bacterium PAO1 is purchased from German DSMZ (preserving numbers：DSM 1707), Pseudomonas fluorescens pf5 is purchased from U.S. American Type culture and protects Tibetan center ATCC (preserving numbers：BAA-477^TM), pseudomonas syringae DC3000 is purchased from U.S. American Type culture and protects Tibetan center ATCC (preserving numbers：BAA-871^TM), pseudomonas putida KT2440 be purchased from German DSMZ (preserving numbers：DSM 6125).Competent escherichia coli cell is prepared according to document report flow before.Pseudomonas aeruginosa, Pseudomonas Bacterium, pseudomonas syringae, pseudomonas putida, the bacterial strain with expression plasmid carry out incubated overnight first, are then diluted to In the LB culture mediums (pseudomonas syringae is in KB medium cultures) of 1.3ml, initial OD₆₀₀Value be about 0.085.According to Every plant of most suitable growth temperature of bacterium is cultivated, and 950rpm cultures start to logarithmic phase, are subsequently adding derivant L- rhamnoses extremely Final concentration of 2.5g L^-1, cell 950rpm cultures 40 minutes at a temperature of 30 DEG C or 37 DEG C.When carrying out competent cell preparation, First bacterium solution 9500rpm room temperatures are centrifuged 30 seconds, supernatant is discarded, the ddH of 1ml is added₂O is carried out resuspended and is centrifuged, and repeats this step Suddenly once.Cell is resuspended in 30 μ l ddH by next step₂In O and DNA is added, carry out adding 1500ngPCR to produce when plasmid is modified Thing, adds when carrying out genomic modification for pseudomonas aeruginosa, Pseudomonas fluorescens, pseudomonas syringae and pseudomonas putida Enter 2.5 μ gPCR products.Electric revolving cup uses the specification of 1mm, electric conversion instrument (Eppendorf 2510electroporator) voltage It is set as 1250V.Electricity recovers 60 minutes cell for 100 minutes or 37 DEG C in 30 DEG C of recoveries after the completion of turning, and is finally coated on Appropriate antibiotic concentration it is upper, pseudomonas aeruginosa, Pseudomonas fluorescens, pseudomonas putida second day can be counted on flat board again Number, pseudomonas syringae needs to count for the 3rd or four day.

Embodiment 1：Build a series of homologous recombination expression plasmids

Using the amino acid sequence of λ Beta or rac RecT as reference, BLAST analyses are from pseudomonad and false unit cell The genome of bacterium bacteriophage, finds the potential albumen with recombination function.Sent out from pseudomonad and pseudomonad bacteriophage Two operators comprising paired exonuclease-recombinase are showed, an operator is from pseudomonas syringae RecTEpsy, this operator has been used successfully to build recombination system；Second operator comes from pseudomonad bacteriophage VB_PaeP_Tr60_Ab31, this operator encodes three albumen:Orf38 (B) contains 177 amino acid, and sequence has with Red β 62% homology；Orf37 (A) contains 195 amino acid, and sequence has 32% homology with Red α；Orf36 (S) contains 148 Individual amino acid, sequence has 54% homology with the single-stranded DNA binding protein SSB of Escherichia coli.

A series of recombination function plasmids are built based on the replicon pBBR1 of wide host, recombination function albumen profit Induced expression is carried out with rhamnose promoter.

The construction method of pBBR1-Rha-red_gba-kan plasmids：PBBR1-Rha-GFP-kan is extracted from Escherichia coli Plasmid, taking 40ul plasmid NdeI digestion 3h, agarose gel electrophoresis 90V carries out race glue, until fragment is run to gel bottom The gel for carrying out linear carrier is reclaimed, and with pSC101-BAD-gba-tet plasmids as template, gba-3/gba-5 is expanded for primer Red_gba fragments, by the culture of GB-dir overnight bacterials and 30 DEG C of switching 1.3ml culture 2h, add 20ul arabinose derivants 37 DEG C of induction 40min, induction is dissolved by pBBR1-Rha-kan linear carriers and red_gba fragments 1350V electric shock corotation In GB05-dir, 37 DEG C of recovery 1h, thalline are centrifuged for 10000rpm and line is applied to the resistant panel of kanamycins 15,37 DEG C of mistakes Night cultivates and chooses monoclonal and identified, correct clone carries out bacterium guarantor.

The construction method of other plasmids is similar to：PBBR1-Rha-plu_gba-kan plasmids are linearly carried by pBBR1-Rha-kan Body is dissolved in GB05-dir with plu_gba fragments electric shock corotation, and arabinose is induced and obtained into the restructuring of line line；pBBR1-Rha- TEpsy-kan plasmids are by pBBR1-Rha-kan linear carriers and TE_PsyFragment electric shock corotation is dissolved in GB05-dir, arabinose Induce and obtained into the restructuring of line line；PBBR1-Rha-pluGam-TEpsy-kan plasmids are linear by pBBR1-Rha-TEpsy-kan Carrier is dissolved in GB05-dir with pluGam fragments electric shock corotation, and arabinose is induced and obtained into the restructuring of line line；pBBR1- Rha-redGam-TEpsy-kan plasmids are dissolved by pBBR1-Rha-TEpsy-kan linear carriers with redGam fragments electric shock corotation In GB05-dir, arabinose is induced and obtained into the restructuring of line line；PBBR1-Rha-BAS-kan plasmids are by pBBR1-Rha-kan Linear carrier is dissolved in GB05-dir with BAS fragments electric shock corotation, and arabinose is induced and obtained into the restructuring of line line；pBBR1- Rha-pluGam-BAS-kan plasmids are dissolved by pBBR1-Rha-BAS-kan linear carriers with pluGam fragments electric shock corotation In GB05-dir, arabinose is induced and obtained into the restructuring of line line；PBBR1-Rha-redGam-BAS-kan plasmids are by pBBR1- Rha-BAS-kan linear carriers are dissolved in GB05-dir with redGam fragments electric shock corotation, and arabinose is induced into line line weight Group is obtained.

Outside host specificity exonuclease and single stranded DNA annealing two genes of albumen, or in host specificity Exonuclease, single stranded DNA annealing albumen and external source or endogenous nucleic acid excision enzyme recBCD suppress outside three genes of albumen, the Three genes or the 4th gene can also be derived from the single-stranded DNA binding protein SSB of bacterial chromosome, or derive from SSB on bacterial plasmid, or from the SSB of bacteriophage, or from the SSB of bacterium prophge.

Embodiment 2：The efficiency comparison of different recombination systems in pseudomonad

By 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 imported into four kinds of false unit cells by electroporated Bacterium (pseudomonas aeruginosa, pseudomonas syringae, Pseudomonas fluorescens and pseudomonas putida).Figure 1A is recombination system efficiency ratio Compared with schematic diagram.Pseudomonas aeruginosa has good upgrowth situation at 37 DEG C and 30 DEG C, it is therefore desirable at a temperature of both Carry out efficiency comparison.Pseudomonas aeruginosa has various resistances to the action of a drug and is readily available stronger resistance mechanism in itself, so Accuracy is calculated after carrying out recombination efficiency relatively in the pseudomonas aeruginosa.

In pseudomonas aeruginosa, Red γ and Plu γ albumen be added in BAS recombination systems can significantly improve it is homologous The accuracy of restructuring, but for pBBR1-Rha-TEpsy-kan plasmids, Plu γ albumen is added just under only being cultivated at 30 DEG C Accuracy (Figure 1B) can be improved.When cultivating for 37 DEG C, pBBR1-Rha-BAS-kan plasmids can work, after adding Red γ albumen The accuracy (Fig. 1 C) of genetic recombination can be significantly improved.Therefore when we carry out genetic modification in pseudomonas aeruginosa, 30 PBBR1-Rha-pluGam-TEpsy-kan plasmids are used under the conditions of DEG C, pBBR1-Rha-RedGam- is used under the conditions of 37 DEG C BAS-kan plasmids.Situation to pseudomonas aeruginosa is similar, and Red γ and Plu γ albumen is added in Pseudomonas fluorescens When on pBBR1-Rha-TEpsy-kan and pBBR1-Rha-BAS-kan plasmids, it is also possible to significantly improve recombination efficiency (Fig. 1 D). But in pseudomonas syringae and pseudomonas putida, Red γ and Plu γ albumen is added to pBBR1-Rha-BAS-kan Recombination efficiency (Fig. 1 E, 1F) can be reduced when on plasmid.

It is final to determine pseudomonad (pseudomonas aeruginosa, pseudomonas syringae, Pseudomonas fluorescens and Pseudomonas putida Bacterium) in efficient recombinant expression plasmid be pBBR1-Rha-BAS-kan plasmids, pBBR1-Rha-redGam-TEpsy-kan matter Grain, pBBR1-Rha-pluGam-TEpsy-kan plasmids, pBBR1-Rha-pluGam-BAS-kan plasmids and pBBR1-Rha- RedGam-BAS-kan plasmids.Wherein S is derived from the single-stranded DNA binding protein of bacteriophage vB_PaeP_Tr60_Ab31 (SSB), Red γ are derived from the suppression albumen of the Escherichia coli nucleic acid excision enzyme recBCD of lambda bacteriophages, and Plu γ come Come from the suppression albumen of the exonuclease recBCD of luminous bacillus (Photorhabdus luminescens) prophge.

Embodiment 3：The optimization of recombination system condition of work is so as to improve recombination efficiency in pseudomonad

According to embodiment 2, the optimal recombination system of every kind of pseudomonad is different, and this several recombination system is different In pseudomonad, in competent cell Optimization of preparation, the optimization of DNA inversion quantities, antibiotic selection concentration optimization, homologous brachium On the basis of degree optimization, recombination efficiency can be improved.

The efficiency that the ability of the electroporated entrance cell of exogenous DNA occurs homologous recombination with DNA molecular has much relations, outward The best period of the electroporated entrance cell of source DNA is exponential phase of growth.It is false single that the present invention determines pseudomonas aeruginosa, stench The growth curve (Fig. 2A BCD) of born of the same parents bacterium, pseudomonas syringae and Pseudomonas fluorescens.Data display pseudomonas aeruginosa, stench The exponential phase of growth of pseudomonad, pseudomonas syringae and Pseudomonas fluorescens starts from 2h, 2h, 3h and 1h respectively.According to survey Fixed growth curve, can analyze the Best Times of conversion.Result display pseudomonas aeruginosa, pseudomonas putida, cloves are false Monad and the optimal transformation time of Pseudomonas fluorescens are respectively 2.5h, 2.5h, 4h and 1.5h (Fig. 2 EFGH).

The present invention detects whether the concentration of conversion DNA can influence genetic recombination efficiency simultaneously.Pseudomonas aeruginosa carries PBBR1-Rha-pluGam-TEpsy-kan plasmids, pseudomonas putida carries pBBR1-Rha-BAS-kan plasmids, and cloves is false single Born of the same parents bacterium carries pBBR1-Rha-BAS-kan plasmids, and Pseudomonas fluorescens carries pBBR1-Rha-redGam-TEpsy-kan plasmids, Convert the PCR primer (gentamicin resistance gene) of quantitative same volume respectively, wherein amount of DNA is 100ng, 250ng, 500ng, The gradient of 1000ng, 1500ng and 2000ng, then counts recombination efficiency.Show false by the detection of this gradient DNA concentration Saturation amount of DNA when monad is recombinated is 1500ng (Fig. 3 ABCD).

The present invention is investigated influence of the homologous arm lengths to homologous recombination efficiency.Pseudomonas aeruginosa carries pBBR1- Rha-pluGam-TEpsy-kan plasmids, pseudomonas putida carries pBBR1-Rha-BAS-kan plasmids, and pseudomonas syringae is taken Band pBBR1-Rha-BAS-kan plasmids, Pseudomonas fluorescens carries pBBR1-Rha-redGam-TEpsy-kan plasmids electricity respectively Hit the PCR primer (gentamicin resistance gene) for being transferred to different length homology arm.Two can be attributed in pseudomonas aeruginosa Type a, type is homologous arm lengths less than 50bp, substantially no recombination function (Fig. 3 E)；Second type is homology arm From 75bp to 150bp, in this length range, recombination efficiency is significantly increased (Fig. 3 E) length.For pseudomonas putida, The most short homologous arm lengths of pseudomonas syringae and Pseudomonas fluorescens are respectively 75bp (Fig. 3 F), 50bp (Fig. 3 G), 100bp (Fig. 3 H).

Embodiment 4：The heredity of genome is carried out using each optimal new recombination system in different pseudomonads Modification

The present invention has been successfully established the optimal recombination system of different pseudomonads and to this series of recombination system Condition of work be optimized, next step carries out genome using a series of this optimal recombination system to different pseudomonads Genetic modification.Short homology arm (50bp-150bp) and homology arm long are led in pseudomonas intermediary not of the same race (more than 150bp) Between homologous recombination, carry out genomic DNA genetic modification.The preferred pBBR1-Rha-pluGam- in pseudomonas aeruginosa BAS-kan plasmids, preferred pBBR1-Rha-RedGam-BAS-kan plasmids, pBBR1-Rha- in pseudomonas syringae RedGam-TEpsy-kan plasmids or pBBR1-Rha-pluGam-TEpsy-kan plasmids, in pseudomonas putida preferably PBBR1-Rha-BAS-kan plasmids, in the preferred pBBR1-Rha-BAS-kan plasmids of Pseudomonas fluorescens or pBBR1-Rha- PluGam-TEpsy-kan plasmids.

The genomic modification of pseudomonas aeruginosa PAO1 includes gene knockout and gene knock-in.Fig. 4 A are shown clpp gene Except process schematic, one section of PCR primer is inserted into by target gene by homologous recombination first, this section of PCR primer includes that celebrating is big mould The resistant gene of element, homology arm (75bp) and locus specificity recombinate enzyme recognition site loxM (lox71-lox66), target gene point It is not：；PhnAB genes, it is the indispensable gene of pyo biosynthesis；LasB genes, it encodes elastoser B； MdoH genes, it encodes an albumen and is related to the biosynthesis of pericentral siphon glucan.After genetic recombination, using 5 ' -3 ' end bacterium colony PCR is verified (Fig. 4 BCD).Carrying out once electricity conversion using pBBR1-Rha-pluGam-TEpsy-kan plasmids can about obtain To 100 or so correct gentamicin resistance clones.But lost using pBBR1-Rha-RedGam-BAS-kan plasmids When passing modification, accuracy will decline.In order to obtain the seamless modification mutant of pseudomonas aeruginosa PAO1, by Cre enzymes Locus specificity restructuring knocks out resistant gene, and finally various mutant are carried out with bacterium colony PCR identifications (Fig. 4 EFG).

ToxA has three domains：Amino terminal domain I includes a recognition site for eukaryotic, middle structure Domain II includes protein delivery function, and carboxy-terminal domains III has a binding site for biology enzyme.On Domain III 578 Position glutamic acid is avtive spot, and this amino acid substitution can be built one plant of bacterial strain of attenuation.The point mutation of ToxA is (578 The codon of position is changed into GAC from GAG, and glutamic acid is changed into aspartic acid) acquisition (Fig. 5 A) can be recombinated by two simple and direct steps. In first step restructuring, by expressing pBBR1-Rha-pluGam-TEpsy-kan plasmids or pBBR1-Rha-RedGam-BAS- Kan plasmids, are transformed into verdigris false single by the PCR primer with gentamicin resistance gene and a reverse selection markers (SacB) Born of the same parents' bacterium cell, homologous recombination is carried out with target gene.Random selection recombinant clone carries out the bacterium colony PCR identification (figures at 3 ' ends and 5 ' ends 5B), correct clone then carries out the functional verification (Fig. 6) of SacB.In the restructuring of second step, by table pBBR1-Rha- PluGam-TEpsy-kan plasmids or pBBR1-Rha-RedGam-BAS-kan plasmids, will be (close 578 comprising point mutation Numeral is changed into GAC from GAG, and glutamic acid is changed into aspartic acid) single stranded DNA be transformed into the mutant for building before, recombinate The bacterial strain of point mutation will be obtained afterwards.Final mutation strain verifies (Fig. 5 C) and sequence verification (Fig. 5 D) by bacterium colony PCR.

In order to extend the range of application of new recombination system, the present invention can also be by inserting promoter (P_apra) mode To activate the biosynthesis pathway of secondary metabolite.By expressing pBBR1-Rha-BAS-kan plasmids, by promoter (P_genta) It is inserted into pseudomonas syringae tomato variety DC3000 (Pseudomonas syringae pv.tomato str.DC3000) (Fig. 7 A).But there is no corresponding compound in HPLC-MS analyses.

In Pseudomonas fluorescens, a kind of mixing sensing kinases for being called RetS can suppress the generation of some antibiotic, Including 2,4- diacetyls phloroglucin, pyrroles's nitro and yellowish green pyocin.Gene is carried out to retS in Pseudomonas fluorescens Inactivation, may improve this plant of biological and ecological methods to prevent plant disease, pests, and erosion ability of bacterium.By expressing pBBR1-Rha-redGam- in Pseudomonas fluorescens TEpsy-kan plasmids, can be knocked out retS genes, and mutant strain is verified (Fig. 7 B) by bacterium colony PCR.

Embodiment 5：The expression of SSB albumen can improve the recombination efficiency of pseudomonas aeruginosa and Escherichia coli

In Escherichia coli, the expression of SSB albumen can significantly improve the weight of pBBR1-Rha-RedGam-BA-kan plasmids Group efficiency, but SSB albumen can not but improve the recombination efficiency (Fig. 8 A) of pBBR1-Rha-BA-kan plasmids, in P. aeruginosa The result (Fig. 8 B) being also similar in bacterium.In pseudomonas syringae, SSB albumen can significantly improve pBBR1-Rha- The recombination efficiency of BA-kan plasmids, but recombination efficiency is reduced on the contrary after adding Red γ albumen, and also SSB will not also be carried Recombination efficiency (Fig. 8 C) high.Result shows that SSB improves recombination efficiency and may have relation with host protein.

Sequence table

<110>Shandong University

<120>New recombination system and its application in pseudomonad

<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 (6)

1. a kind of new recombination system in pseudomonad, the recombination system is made up of a series of homologous recombination system expression plasmids, It is characterized in that：The expression plasmid expresses an operator under inducible promoters, and the operator is removed comprising host specific Property exonuclease and single stranded DNA annealing two genes of albumen outside, the 3rd gene is derived from Escherichia coli lambda and bites The Red γ of thalline, or from the Plu γ of luminous bacillus (Photorhabdus luminescens) prophge (Plu2934), or other external sources exonuclease recBCD suppress albumen；Or, the operator is removed comprising host specific Property exonuclease, single stranded DNA annealing albumen and external source or endogenous nucleic acid excision enzyme recBCD suppress outside three genes of albumen, 4th gene is derived from the single-stranded DNA binding protein SSB of bacterial chromosome, or the SSB on bacterial plasmid, or The SSB of bacteriophage is derived from, or from the SSB of bacterium prophge.

2. new recombination system in pseudomonad according to claim 1, it is characterised in that：Series in the recombination system Homologous recombination system expression plasmid includes pBBR1 replication orgins, kalamycin resistance gene and rhamnose inducible promoter Recombination system；Its name is respectively pBBR1-Rha-red_gba-kan, and nucleotide sequence is as shown in SEQ ID No.23； PBBR1-Rha-plu_gba-kan, nucleotide sequence is as shown in SEQ ID No.24；PBBR1-Rha-TEpsy-kan, nucleotides Sequence is as shown in SEQ ID No.25；PBBR1-Rha-pluGam-TEpsy-kan, nucleotide sequence such as SEQ ID No.26 institutes Show；PBBR1-Rha-redGam-TEpsy-kan, nucleotide sequence is as shown in SEQ ID No.27；PBBR1-Rha-BAS-kan, Nucleotide sequence is as shown in SEQ ID No.28；PBBR1-Rha-pluGam-BAS-kan, nucleotide sequence such as SEQ ID Shown in No.29；PBBR1-Rha-redGam-BAS-kan, nucleotide sequence is as shown in SEQ ID No.30；Wherein, RecTEpsy The RecTEpsy in the prophge of pseudomonas syringae (Pseudomonas syringae) is derived from, B is that single stranded DNA is moved back Fiery albumen or be recombinase, A is 5 ' -3 ' double-stranded DNA exonucleases, and S is derived from pseudomonas aeruginosa The single-stranded DNA binding protein (SSB) of (Pseudomonas aeruginosa) bacteriophage vB_PaeP_Tr60_Ab31, Red γ are From the suppression albumen of the Escherichia coli nucleic acid excision enzyme recBCD of lambda bacteriophages, Plu γ are derived from luminous bacillus The suppression albumen of the exonuclease recBCD of (Photorhabdus luminescens) prophge.

3. new recombination system in pseudomonad according to claim 2, it is characterised in that the series in the recombination system Homologous recombination system expression plasmid is：PBBR1-Rha-BAS-kan, pBBR1-Rha-pluGam-TEpsy-kan, pBBR1- Rha-redGam-TEpsy-kan, pBBR1-Rha-redGam-BAS-kan or pBBR1-Rha-pluGam-BAS-kan.

4. new recombination system in the pseudomonad according to claim 1,2 or 3, it is characterised in that the pseudomonad It is pseudomonas aeruginosa, pseudomonas syringae, Pseudomonas fluorescens or/and pseudomonas putida.

5. new recombination system leads short homology arm and length in pseudomonas intermediary not of the same race in pseudomonad described in claim 3 Homologous recombination between homology arm carries out the application in genomic DNA or plasmid genetic modification.

6. application according to claim 5, it is characterised in that：

The short homology arm refers to the homology arm of 50bp-150bp, and the homology arm long refers to the homology arm more than 150bp；It is described It is false single in verdigris when mediating the homologous recombination between short homology arm and homology arm long and carrying out genomic DNA or plasmid genetic modification PBBR1-Rha-redGam-BAS-kan is selected in born of the same parents bacterium (Pseudomonas aeruginosa), in pseudomonas syringae PBBR1-Rha-redGam-BAS-kan, pBBR1-Rha-redGam-TEpsy-kan are selected in (Pseudomonas syringae) Or pBBR1-Rha-pluGam-TEpsy-kan, select pBBR1-Rha- in pseudomonas putida (Pseudomonas putida) BAS-kan, pBBR1-Rha-BAS-kan or pBBR1- is selected in Pseudomonas fluorescens (Pseudomonas fluorescens) Rha-pluGam-TEpsy-kan。