CN102559729B - Expression plasmid for integrating and evolving chromosome - Google Patents

Expression plasmid for integrating and evolving chromosome Download PDF

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CN102559729B
CN102559729B CN 201210060042 CN201210060042A CN102559729B CN 102559729 B CN102559729 B CN 102559729B CN 201210060042 CN201210060042 CN 201210060042 CN 201210060042 A CN201210060042 A CN 201210060042A CN 102559729 B CN102559729 B CN 102559729B
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gene
resistance
expression plasmid
resistance screening
chromosome
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CN102559729A (en
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刘建忠
黄明涛
崔艳艳
陈韵妍
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Sun Yat Sen University
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Abstract

The invention discloses an expression plasmid for integrating and evolving a chromosome, and the expression plasmid comprises two FRT sites, a resistance screening gene alpha, a replicon, a bacteriophage integration site (attP), a promoter (P), a multiple cloning site (MCS) behind the promoter, a terminator (TTR), a resistance screening gene beta for inducing the evolutionary process of the chromosome, and same segments A1 and A2 of two heterogenous nucleotide sequences connected with two sides of the promoter and the resistance screening gene beta, wherein the resistance screening gene alpha and the resistance screening gene beta are different resistance genes. According to the expression plasmid, a target gene can be inserted into the chromosome of engineering bacteria by virtue of simple plasmid transformation, the copy number of the target gene on the chromosome can be improved by virtue of chemical induction, the expression plasmid is simple to operate, the production efficiency of a target product can be greatly improved, and the expression plasmid is suitable for industrial production. Furthermore, the target gene is integrated onto the chromosome, so that the built engineering bacteria can not be lost in the process of passage, and is high in genetic stability and stable in production performance.

Description

A kind of expression plasmid for chromosomal integration, evolution
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of expression plasmid for chromosomal integration, evolution, with and construction process.
Background technology
Utilize genetically engineered microorganism to produce useful matter and become the new model that material is made.At present, usually adopt the plasmid expression system that contains goal gene to build engineered microbes.But this plasmid expression system often has and has the metabolism burden that plasmid is easily lost, existence unstable and plasmid will increase again Host Strains.More seriously plasmid often contains again the antibiotics resistance selection markers, and this will have a strong impact on problem of environmental pollution, causes that in environment, antibiotic resistant bacteria spreads unchecked, thereby has limited the process of industrialization of the engineering bacteria that contains plasmid.
In order to solve the defects of plasmid expression system, it is a very effective method that goal gene is incorporated on the microbial staining body.For this reason, many scientists have researched and developed many plasmids and come integrator gene to karyomit(e).A series of conditions of having developed the Haldimann and Wanner of Purdue Univ-West Lafayette USA (2001) copy to integrate regulates (CRIM) plasmid, utilize this plasmid can be with the goal gene that is connected in advance on plasmid, by simple Plasmid Transformation, single copy ground is incorporated on escherichia coli chromosome specifically (Haldimann on the phage integration site, A., Wanner, B.L.Journal of Bacteriology.2001,183:6384-6393).But after utilizing these plasmid integration genes to the colibacillary karyomit(e), antibiotics resistance selection markers and replicon on plasmid still are retained on karyomit(e).Constructed engineering bacteria still exists the antibiotics resistance selection markers to cause the problem that in environment, resistant organism spreads unchecked.for overcoming their plasmid defective, Taiwan Chiang et al. has and has developed a series of plasmids for gene integration on the basis of their work, using these plasmids equally can be with the goal gene that is connected in advance on plasmid, by simple Plasmid Transformation, single copy ground is incorporated on escherichia coli chromosome specifically on the phage integration site, and resulting engineering bacteria does not contain antibiotics resistance selection markers and replicon, can be used for suitability for industrialized production (Chiang, C.-J., Chen, P.T., Chao, Y.P.Biotechnology Bioengineering 2008, 101:985-995).But the plasmid of above-mentioned 2 team all can only be incorporated into the goal gene list on colibacillary karyomit(e) with copying, and often needs to integrate the gene of multiple copied in real work with the expression level of raising gene, thereby improves the throughput of microorganism.for this reason, the Tyod et al. of Massachusetts Institute Technology has developed a kind of plasmid pTGD that improves the integrator gene copy number, utilize λ InCh genome conformity technology, after being incorporated into the goal gene on plasmid on escherichia coli chromosome, because of carried on plasmid antibiotics resistance gene (with the goal gene tandem together), can utilize so-called chemical induction chromosome evolution technology, improve the copy number (Tyo of goal gene on karyomit(e), K.E.J., Ajikumar, P.K., Stephanopoulos, G.Nature Biotechnology 2009, 27:760-765).Although the copy number of their resulting engineering bacteria gene has obtained increase because of the chemical induction chromosome evolution, but the gene integration technology that they use is λ InCh genome conformity technology, need 4 steps, more complicated, be unfavorable for industrial application, and engineering bacteria still contains the antibiotics resistance selection markers, is not suitable for suitability for industrialized production.
Summary of the invention
For existing deficiency in prior art, the purpose of this invention is to provide a kind of expression plasmid for chromosomal integration, evolution.This expression plasmid can insert goal gene in the karyomit(e) of engineering bacteria by direct Plasmid Transformation, and can improve the copy number of goal gene on karyomit(e) by chemical induction.
The technical solution adopted in the present invention is:
A kind of expression plasmid for chromosomal integration, evolution, the multiple clone site (MCS), terminator (TTR), the resistance screening gene β that is used for the induced chromosome evolutionary process, and nucleotide sequence same clip A1 and the A2 of 2 allos being connected with resistance screening gene β both sides of promotor that contain 2 FRT sites (can be identified by the FLP recombinase), resistance screening gene a, replicon, phage integration site (attP), promotor (P) and back thereof, resistance screening gene a and resistance screening gene β are different resistant genes.
Preferably, the structure of described plasmid is: the side in two FRT sites contain successively in the direction of the clock Segment A 1, promotor (P) and back thereof multiple clone site (MCS), terminator (TTR), be used for resistance screening gene β, Segment A 2, the phage integration site (attP) of induced chromosome evolutionary process, the opposite side in two FRT sites contains resistance screening gene a and replicon (as shown in Figure 1).
Preferably, described resistance screening gene a is kalamycin resistance gene (kan).
Preferably, described replicon is colibacillary condition replicon (ori R γ).
Preferably, described phage integration site (attP) is attP HK, attP P21, attP λ, attP φ 80And attP P22In any.
Preferably, described promotor is P T5, P T7, P tac, P trc, P lac, P BAD, P Ltet, P LlacEtc. any in promotor.
Preferably, described resistance screening gene β for the induced chromosome evolutionary process is any of triclosan resistant gene (fabI), chloramphenicol resistance gene (cat), ampicillin resistance gene (b/a), gentamicin resistant gene (gen), trimethoprim resistant gene (dhfr), spectinomycin resistance gene (aadA), streptomycin resistance gene (aadA), tetracycline resistance gene (tetA), lactobacillin resistant gene.
Further preferred; described resistance screening gene β for the induced chromosome evolutionary process is triclosan resistant gene (fabI); it is the gene of the fgs encoder alkene acyl of intestinal bacteria-acetyl-carrier protein reductase enzyme own due to the fabI gene; so the genetic engineering bacterium that the conversion of this plasmid obtains can not cause that in environment, antibiotic resistant bacteria spreads unchecked, and is suitable for suitability for industrialized production.
Preferably, described A1, A2 fragment are the allos fragment that comes from except intestinal bacteria, and the homology in intestinal bacteria is low and do not express, and size is 800～1200kbp, can come from any biology except intestinal bacteria.
Preferably, the Host Strains of the above expression plasmid conversion is intestinal bacteria (Escherichia coli).
Beneficial effect of the present invention is:
(1) using plasmid of the present invention can be by Plasmid Transformation with gene integration specific phage integration site to the escherichia coli chromosome, then carry out chromosome evolution under chemical induction, to improve the copy number of integrator gene on karyomit(e), thereby build the even recombination bacillus coli of antibiotic-free resistance screening mark, multiple copied number of plasmid-free, to produce useful matter;
(2) the gene integration step of application plasmid of the present invention is simple, efficient is high.Constructed engineering bacteria can not lost in the process that goes down to posterity because goal gene is incorporated on karyomit(e), and genetic stability is high, thereby causes production performance stable;
(3) use the constructed engineering bacteria of triclosan induced chromosome evolution plasmid pXKF3T5b and do not contain the antibiotics resistance mark, can not cause the problem that in environment, antibiotic resistant bacteria spreads unchecked, be suitable for suitability for industrialized production, the product safety of production.
Description of drawings
Fig. 1 is the physical map that the present invention is used for the expression plasmid of chromosomal integration, evolution;
Fig. 2 is the physical map that embodiment 1 is used for the plasmid pXKF3T5b of chromosomal integration, the evolution of paraxin induced chromosome;
Fig. 3 is the physical map that embodiment 2 is used for the plasmid pXKF3T5b of gene integration, the evolution of triclosan induced chromosome.
Embodiment
The present invention is further illustrated below in conjunction with embodiment, but be not limited to this.
All adopts the method for routine the molecular biology experiment technology that adopts in following examples comprises that pcr amplification, plasmid extraction, Plasmid Transformation, DNA fragmentation connect, enzyme is cut, gel electrophoresis etc., specifically can be referring to " molecular cloning experiment guide " (third edition) (Sambrook J, Russell DW, Janssen K, the yellow training hall of Argentine J. waits to be translated, 2002, Beijing: Science Press).
In following examples, primer used sees Table 1.
Table 1
Figure BDA0000141506640000051
Embodiment 1
The expression plasmid pXKC3T5b that is used for chromosomal integration, the evolution of paraxin induced chromosome
X in plasmid pXKC3T5b represents to contain different phage integration sites, comprises the HK (attP for the HK022 phage HK), for the P21 (attP of P21 phage P21), for the φ 80 (attP of φ 80 phages φ 80), for the λ (attP of lambda particles phage λ), for the P22 (attP of P22 phage P22).
as shown in Figure 2, plasmid pXKC3T5b contains 2 FRT sites (can be identified by the FLP recombinase), kalamycin resistance gene (kan), colibacillary condition replicon (ori R γ), phage integration site (attP), the multiple clone site of T5 promotor and back thereof (MCS) and terminator (TTR), be connected to the chloramphenicol resistance gene (cat) that induced chromosome is evolved that is used for after multiple clone site, be connected to 2 nucleotide sequence same clip A1 and A2 that come from Corynebacterium glutamicum cgl1740 gene of promotor and chloramphenicol resistance gene (cat) both sides, this fragment homology in intestinal bacteria is low, and can not express.
Its construction process is as follows:
1, build pHK-Kan
At first with reference to Taiwan Chiang et al. method (Chiang, C.-J., Chen, P.T., Chao, Y.P.Biotechnology Bioengineering 2008,101:985-995) structure pHK-Kan.With primer pair RCWF (SEQ ID NO:3), (professor Wanner of Purdue Univ-West Lafayette USA is so kind as to give RCWR (SEQ ID NO:4) from pAH144, referring to Haldimann, A., Wanner, B.L.Journal of Bacteriology.2001,183:6384-6393) upper amplification its multiple clone site (MCS) and attP HKThe site, (professor Wanner of Purdue Univ-West Lafayette USA is so kind as to give primer pair RCNF (SEQ ID NO:5), RCNR (SEQ ID NO:6) amplification pKD3, referring to Datsenko, K.A., Wanner, B.L PNAS.2000,97:6640-6645) on do not comprise the rest part of cat gene.Both PCR products are connected to each other after cutting with Pst I+Xho I enzyme, just build and obtain pHK-Amp.Again the kan gene between the upper Sph I-Not I restriction enzyme site of pAH120 is downcut, replace the bla gene (Amp between the upper Sph I-Not I restriction enzyme site of pHK-Amp r), build and obtain pHK-Kan.
2, build pHKKC3T5b
With primer pair CG1F (SEQ ID NO:7), CG1R (SEQ ID NO:8) cgl1740 gene amplification upstream homology arm sequence A 1 from the C.glutamicum genome, and cut this fragment (EcoT22 I and Pst I are isocaudarner) with EcoT22I+Pst I enzyme, forward inserts the Pst I site of pHK-Kan, builds to obtain pHK-KanA1.With primer pair CG2F (SEQ ID NO:9), CG2R (the SEQ ID NO:10) restriction enzyme site that amplification downstream, same position homology arm sequence A 2 is additionally added except end from the C.glutamicum genome, A2 sequence and A1 are in full accord), and cut this fragment (EcoR I and Mun I are isocaudarner) with EcoR I+Mun I enzyme, forward inserts the EcoR I site of pHK-KanA1, builds to obtain pHK-KanA2.
Increase from pKD3 not with the cat gene in FRT site with primer pair Cm1 (SEQ ID NO:11), Cm3 (SEQ ID NO:12), and cut with restriction endonuclease EcoR I+Mun I enzyme the cat gene that PCR obtains, forward inserts the EcoR I site of pHK-KanA2, builds to obtain pHK-KanC3.PHK-KanC3 cuts with Sph I enzyme, and the Primestar archaeal dna polymerase fills end from connecting, and eliminates SphI, obtains plasmid pHK-KanC3b.Again the T5 promotor in p18S-Q3Fb and terminator are downcut with Mlu I+ApaL I, replace the sequence between Mlu I-ApaL I site in pHK-KanC3b, namely obtain pHKKC3T5b (SEQ ID NO:1).
3, build the plasmid that contains other phage integration sites
With primer AH1 (SEQ ID NO:13), AH2 (SEQ ID NO:14) amplification phage site attP from pAH81, pAH120, pAH153 and pAH154 respectively P21, attP λ, attP φ 80And attP P22, and be connected in the T carrier.Check order errorless after, then with Sac I+Xho I, they are downcut, replace the attP on pHKKC3T5b HK, correspondence obtains containing plasmid pP21KC3T5b, pP22KC3T5b, p λ KC3T5b, the p φ 80KC3T5b of corresponding integration site.
Embodiment 2
The plasmid pXKF3T5b that is used for chromosomal integration, the evolution of triclosan induced chromosome
Chloramphenicol resistance gene cat in the plasmid pXKC3T5b of embodiment 1 is replaced into intestinal bacteria fabI gene (triclosan resistant gene), can obtains the plasmid pXKF3T5b that the triclosan induced chromosome is evolved, its structure as shown in Figure 3.
Its construction process is as follows:
1, at first use primers F abF (SEQ ID NO:15) and FabR (SEQ ID NO:16) pcr amplification gene fabI fragment from genome of E.coli, be connected in pMD18-T simple and obtain pMD-fabI.With primer CG1F (SEQ ID NO:7) and CG2R (SEQ ID NO:11) pcr amplification downstream homology arm sequence A 2 (EcoT22I, Mun I and Sac II restriction enzyme site are contained respectively in front and back) again from the Corynebacterium glutamicum gene group, be connected on the pMD18-T carrier, obtain pMD-A2.
2, cut pMD-A2 with Pst I/EcoT22I enzyme, gel reclaims and contains the A2 fragment, is connected to the EcoT22I enzyme and cuts the linearized vector that pMD-fabI obtains, and obtains pMD-A2-FabI.Cut pMD-A2-FabI with ApaL I/SacII enzyme, recovery contains the A2-FabI fragment, is connected to same enzyme and cuts on the pXKC3T5b of processing, just can obtain pXKF3T5b.Wherein the sequence of pP21KF3T5b is as shown in SEQ ID NO:2.
Only for introducing preferred case of the present invention, to those skilled in the art, any apparent changes and improvements of carrying out in the scope that does not deviate from spirit of the present invention all should be regarded as a part of the present invention to above embodiment.For example: the promotor in plasmid of the present invention is except available P T5Can also select P outward, T7, P tac, P trc, P lac, P BAD, P Ltet, P LlacIn any, all drop in protection scope of the present invention; 2 nucleotide sequence same clip A1 on plasmid and A2 are except being to come from the L-glutamic acid bacillus cgl1740 of side gene, can also be from any biology except intestinal bacteria, as long as in intestinal bacteria homology low and do not express, about big or small 1kbp, all drop in protection scope of the present invention; be connected to and be used for resistant gene that induced chromosome evolves except being chloramphenicol resistance gene (cat) and triclosan resistant gene (fabI) after multiple clone site, can also be other resistant genes, as ampicillin resistance gene (bla), gentamicin resistant gene (gen), trimethoprim resistant gene (dhfr), spectinomycin resistance gene (aadA), streptomycin resistance gene (aadA), tetracycline resistance gene (tetA), lactobacillin resistant gene etc., all drop in protection scope of the present invention.
The below illustrates the application of expression plasmid of the present invention take the engineering colon bacillus that builds plasmid-free, produce Lyeopene as example.
With intestinal bacteria E.coli BW25113 (Δ gdhA Δ aceF, P T5-dxs) for the bacterium that sets out is the structure of product lycopene engineering bacteria of bacterium of setting out, comprise the following steps:
(1) (professor Wanner of Purdue Univ-West Lafayette USA is so kind as to give will to express the helper plasmid pAH121 of intergrase, referring to Haldimann, A., Wanner, B.L.Journal of Bacteriology.2001,183:6384-6393) be transformed into intestinal bacteria E.coli BW25113 (Δ gdhA Δ aceF, P T5-obtain intestinal bacteria E.coliBW25113 (Δ gdhA Δ aceF, P in dxs) T5-dxs, pAH121).
(2) Hind III enzyme is cut pB-crtEIBipi (expression vector that contains the Lyeopene synthetic gene) and is filled, and then cut with EcoR I enzyme, gel reclaims the Lyeopene synthetic gene crtEIBipi fragment of 4329bp, be connected on the pP21KF3T5b carrier (SEQ ID NO:2) after EcoR I/BamH III enzyme is cut, obtain pP21KF3T5b-crtEIBipi.
(3) gene integration: pP21KF3T5b-crtEIBipi is transformed into intestinal bacteria E.coli BW25113 (Δ gdhA Δ aceF, P T5-dxs, pAH121): the E.coli BW25113 (Δ gdhA Δ aceF, the P that 5 μ l pP21KF3T5b-crtEIBipi are joined 100 μ l T5-dxs, pAH121) in competent cell, placed on ice 30～50 minutes; 42 ℃ of thermal shocks 90 seconds are put into 3～5 minutes on ice at once; Add LB nutrient solution (containing 0.5%KAc) 600 μ l, 37 ℃ of 225rpm shaking tables were cultivated approximately 45～60 minutes, then continued to cultivate 30 minutes in 42 ℃ of shaking baths; Centrifugal, abandon supernatant liquor to surplus approximately 200 μ l, sterilization rifle head piping and druming thalline is extremely evenly; Spreading rod is applied to LB flat board (containing 0.5%KAc, 1 μ M triclosan and kantlex 25 μ g/ml), is inverted overnight incubation for 37 ℃.
(4) removal of helper plasmid pAH121: select red bacterium colony, access in the test tube of the LB liquid nutrient medium that contains 1 μ M triclosan and 0.5%KAc 42 ℃ of overnight incubation.Dip the cell of a small amount of above-mentioned overnight incubation with transfering loop, rule on the flat board of the LB that contains 1 μ M triclosan and 0.5%KAc and separate, in 37 ℃ of overnight incubation.The red single bacterium colony dibbling simultaneously that grows to the flat board of the LB that contains 1 μ M triclosan+0.5%KAc, 100 μ g/mL penbritin+0.5%KAc, in 37 ℃ of overnight incubation, is only selected and contained the red bacterium colony of growing on the flat board of triclosan.
(5) removal of kalamycin resistance gene: only select and containing the red bacterium colony of growing on the flat board of triclosan, preparation chemoreception attitude cell, then (professor Wanner of Purdue Univ-West Lafayette USA is so kind as to give with plasmid pCP20, referring to Datsenko, K.A., Wanner, B.L.PNAS.2000,97:6640-6645) be transformed in the chemoreception attitude cell of preparation, be coated on the LB flat board of 1 μ M triclosan+100 μ g/mL penbritin+0.5%KAc, in 30 ℃ of overnight incubation.The red bacterium colony of picking accesses in the test tube of the LB liquid nutrient medium that contains 1 μ M triclosan and 0.5%KAc, cultivates 6h in 30 ℃ of shaking tables, then continues overnight incubation at 42 ℃.Dip the cell of a small amount of above-mentioned overnight incubation with transfering loop, rule on the flat board of the LB that contains 1 μ M triclosan and 0.5%KAc and separate, in 37 ℃ of overnight incubation.With the red single bacterium colony dibbling simultaneously that grows to the flat board of the LB that contains 1 μ M triclosan+0.5%KAc, 25 μ g/mL kantlex+0.5%KAc, 100 μ g/mL penbritin+0.5%KAc, in 37 ℃ of overnight incubation.Only integrate exactly bacterium containing the red bacterium colony of growing on the flat board of triclosan.
(6) the triclosan induced chromosome is evolved: only select and containing the red bacterium colony of growing on the flat board of triclosan, be inoculated in the test tube of LB (the containing 0.5%KAc) liquid nutrient medium that contains 1 μ M triclosan 37 ℃ of cultivation 24h.Then be transferred in the test tube that contains 2 μ M triclosan LB (containing 0.5%KAc) liquid nutrient mediums, cultivate 24h for 37 ℃, the residue bacterial classification prepares the glycerine kind in-80 ℃ of preservations.The bacterial classification of anti-2 μ M triclosans is continued to be transferred in the test tube that contains 4 μ M triclosan LB (containing 0.5%KAc) liquid nutrient mediums, cultivate 24h for 37 ℃, the residue bacterial classification prepares the glycerine kind in-80 ℃ of preservations.Repeat above-mentioned steps, until till triclosan concentration is 16 μ M.
(7) fermentation of evolution bacterium screening: with the bacterial classification of above-mentioned tolerance different concns triclosan, receive respectively in the test tube of LB (containing 0.5%KAc) liquid nutrient medium of respective concentration triclosan, cultivate 12h for 37 ℃.Then be transferred to 50mL fermention medium (g/L: peptone 12.0, yeast extract 24.0, K is housed 2HPO 411.4, KH 2PO 41.7 KAc 5.0, MgSO 47H 2O 1.0, sucrose 5.0, and ammonium oxalate 10mM, tween-80 2.0, pH 7.0) in, in 37 ℃, 250 rev/mins shaker fermentation 48h.Sampling and measuring cell concn OD during fermentation ends 600Taking a morsel, (approximately 500 μ L, be designated as v to bacterium liquid 1), the centrifugal 5min of 10000rpm under room temperature, remove supernatant, add 500 μ L distilled water washs, the centrifugal 5min of 10000rpm under room temperature, remove supernatant, precipitation is resuspended in～1mL acetone in, repeatedly with the piping and druming of rifle head, extract is incubated 15min in 55 ℃ of water-baths after, the centrifugal 5min of 10000rpm under room temperature gets supernatant liquor, also uses the acetone constant volume to certain volume v 2Take acetone as contrast, utilize the light absorption value of spectrophotometer measurement extract under 472nm, be calculated as follows the concentration of Lyeopene:
Lycopene concentration in fermented liquid (mg/L)=OD 472* 4.418 * v 2/ v 1
The content of Lyeopene in thalline (mg/g dry mycelium)=OD 472* 4.418 * v 2/ (v 1* c)
(8) choose engineering bacteria that in thalline, content of lycopene is the highest and be the engineering colon bacillus that plasmid-free, antibiotic-free resistance screening mark produce Lyeopene.
This shows, after plasmid integration of the present invention is in the Host Strains, by chemical induction chromosome evolution technology, the copy number of goal gene can be evolved to required copy number, obtain the genetic engineering bacterium of plasmid-free, multiple copied, and simple to operate, be suitable for the suitability for industrialized production useful matter (as Lyeopene, ubiquinone 10, shikimic acid, zeaxanthin and isopentene compounds etc.).
＜110〉Zhongshan University
＜120〉a kind of expression plasmid for chromosomal integration, evolution
<130>
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 7171
<212> DNA
＜213〉artificial sequence
<400> 1
agattgcagc attacacgtc ttgagcgatt gtgtaggctg gagctgcttc gaagttccta 60
tactttctag agaataggaa cttcggaata ggaacttcat ttactgcatc ctgtacttcg 120
caatgttgac ctcaaatatc ggaagtacac cgacgtattt cccggtgatc accgcacagg 180
aacttcgtgc gcataacgac aagctcttta gccctgagtt catcaaaaac atgctaaagt 240
atctctggga tcgcgacagc ggtgctggac tcagagctgc atcgggtttc cgcaacgtca 300
tgctcaaatc gggtcgccac attgatattc aacgcctcaa tgaaaaacag ctctttgttg 360
gactcaagcg cctgcttggt ctcctcggtc accagattct tgaatttgat cgtctctctg 420
gtgatgatgc ccatgttgat actaacgagg atgtacttga tctcattgcc tacaacgtct 480
cagacgtggt gggcaccaga ctgctcgctg aggacccggt gtactccggc tctttcgatc 540
tgcgggcagg tctactgagc acctacccag agactgtttt tgatcatgat ggtactttcc 600
gtcagccatc cacgcagatg cgtaaagatc gcctaacgat taatacctca tcagctcagt 660
tcgcagcgcg tattttggcg ccatatcgcc cactccgcga tgtccctgat gcgattggcg 720
acatgccggt ggtgtcttac ttgtacccgg atgcagcagt cgccgaagca acaggtcaaa 780
aacaagtcaa cgtgcttgat gagtcaaaga agttcttcta tgacaacatc accgacccgg 840
aagcacgtgc tgcctttgat gaggtctttg ctttttacgc tgatattgag ggtcgcaact 900
tcaacagtca caatgaggtt attgataccc agattaacca attacgtgct tatctcaacc 960
aggttgtcgc attcgatgca gctgggtatg cgctctatga tgtacgtaca cgttttgagc 1020
agatcttccc caaggatcgc agctacatca acgatgctac ggatatgacc cctcgcgcag 1080
tatcgagctt tgacgatctg gttgcactct gtgatgatat tcgcggtgta cttgatcgag 1140
gtttagagat ctcatctccg aatcatcatg agatggtgga tgctatgcgc aagcagctgc 1200
actatattca ggcattttac cgtgcctggg gacccattca acgccgcttc aatgacgctg 1260
acccagcggt gacccatccg catctcacag tgatctaccc accgctcacc cctgcatccg 1320
cagagaaatt caacaagatc acctcagtcg ctgctgtgag caagcgcacg cgttcgagaa 1380
atcataaaaa atttatttgc tttgtgagcg gataacaatt ataatagatt caattgtgag 1440
cggataacaa tttcacacag aattcattaa agaggagaaa ttaactatga gaggatcgca 1500
tcaccatcac catcacggat ccgcatgcga gctcggtacc ccgggtcgac ctgcagccaa 1560
gcttaattag ctgagcttgg actcctgttg atagatccag taatgacctc agaactccat 1620
ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc 1680
tagcttggcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata 1740
taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt 1800
tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt 1860
aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa 1920
tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt 1980
tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga 2040
ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg 2100
tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa 2160
tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc 2220
ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc 2280
gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt 2340
acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg 2400
cccttaaacg cctggggtaa tgactctcta gcttgaggca tcaaataaaa cgaaaggctc 2460
agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta 2520
ggacaaatcc gccctctaga gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg 2580
acacatgcag ctcccgtgca ccgcctacct gtgacggaag atcacttcgc agaataaata 2640
aatcctggtg tccctgttga taccgggaag ccctgggcca acttttggcg aaaatgagac 2700
gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc 2760
gtattttttg agttgtcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc 2820
actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt 2880
cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta 2940
aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc 3000
ctgatgaatg ctcatccgga attacgtatg gcaatgaaag acggtgagct ggtgatatgg 3060
gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc 3120
tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg 3180
tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc 3240
tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac 3300
ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg 3360
ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt 3420
aatgaattac aacagtactg cgatgagtgg cagggcgggc aattccctgt acttcgcaat 3480
gttgacctca aatatcggaa gtacaccgac gtatttcccg gtgatcaccg cacaggaact 3540
tcgtgcgcat aacgacaagc tctttagccc tgagttcatc aaaaacatgc taaagtatct 3600
ctgggatcgc gacagcggtg ctggactcag agctgcatcg ggtttccgca acgtcatgct 3660
caaatcgggt cgccacattg atattcaacg cctcaatgaa aaacagctct ttgttggact 3720
caagcgcctg cttggtctcc tcggtcacca gattcttgaa tttgatcgtc tctctggtga 3780
tgatgcccat gttgatacta acgaggatgt acttgatctc attgcctaca acgtctcaga 3840
cgtggtgggc accagactgc tcgctgagga cccggtgtac tccggctctt tcgatctgcg 3900
ggcaggtcta ctgagcacct acccagagac tgtttttgat catgatggta ctttccgtca 3960
gccatccacg cagatgcgta aagatcgcct aacgattaat acctcatcag ctcagttcgc 4020
agcgcgtatt ttggcgccat atcgcccact ccgcgatgtc cctgatgcga ttggcgacat 4080
gccggtggtg tcttacttgt acccggatgc agcagtcgcc gaagcaacag gtcaaaaaca 4140
agtcaacgtg cttgatgagt caaagaagtt cttctatgac aacatcaccg acccggaagc 4200
acgtgctgcc tttgatgagg tctttgcttt ttacgctgat attgagggtc gcaacttcaa 4260
cagtcacaat gaggttattg atacccagat taaccaatta cgtgcttatc tcaaccaggt 4320
tgtcgcattc gatgcagctg ggtatgcgct ctatgatgta cgtacacgtt ttgagcagat 4380
cttccccaag gatcgcagct acatcaacga tgctacggat atgacccctc gcgcagtatc 4440
gagctttgac gatctggttg cactctgtga tgatattcgc ggtgtacttg atcgaggttt 4500
agagatctca tctccgaatc atcatgagat ggtggatgct atgcgcaagc agctgcacta 4560
tattcaggca ttttaccgtg cctggggacc cattcaacgc cgcttcaatg acgctgaccc 4620
agcggtgacc catccgcatc tcacagtgat ctacccaccg ctcacccctg catccgcaga 4680
gaaattcaac aagatcacct cagtcgctgc tgtgagcaag cgcccgcgga cccaattctc 4740
atgtttgaca gcttatcact gatcagtgaa ttaatggcga tgacgcatcc tcacgataat 4800
atccgggtag gcgcaatcac tttcgtctct actccgttac aaagcgaggc tgggtatttc 4860
ccggcctttc tgttatccga aatccactga aagcacagcg gctggctgag gagataaata 4920
ataaacgagg ggctgtatgc acaaagcatc ttctgttgag ttaagaacga gtatcgagat 4980
ggcacatagc cttgctcaaa ttggaatcag gtttgtgcca ataccagtag aaacagacga 5040
agaagctagc taatgctctg tctcaggtca ctaatactat ctaagtagtt gattcatagt 5100
gactggatat gttgcgtttt gtcgcattat gtagtctatc atttaaccac agattagtgt 5160
aatgcgatga tttttaagtg attaatgtta ttttgtcatc ctttaggtga ataagttgta 5220
tatttaaaat ctctttaatt atcagtaaat taatgtaagt aggtcattat tagtcaaaat 5280
aaaatcattt gtcgatttca attttgtccc atggctaatt cccatgtcag ccgtctcgag 5340
ttctgcgaag tgatcttccg tcacaggtag gcgcgccgaa gttcctatac tttctagaga 5400
ataggaactt cggaatagga actaaggagg atattcatat ggaccatggc taattcccat 5460
gtcagccgtt aagtgttcct gtgtcactca aaattgcttt gagaggctct aagggcttct 5520
cagtgcgtta catccctggc ttgttgtcca caaccgttaa accttaaaag ctttaaaagc 5580
cttatatatt cttttttttc ttataaaact taaaacctta gaggctattt aagttgctga 5640
tttatattaa ttttattgtt caaacatgag agcttagtac gtgaaacatg agagcttagt 5700
acgttagcca tgagagctta gtacgttagc catgagggtt tagttcgtta aacatgagag 5760
cttagtacgt taaacatgag agcttagtac gtgaaacatg agagcttagt acgtactatc 5820
aacaggttga actgctgatc ttcagatcct ctacgccgga cgcatcgtgg ccggatcttg 5880
cggccgcaaa aattaaaaat gaagttttga cggtatcgaa ccccagagtc ccgctcagaa 5940
gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta 6000
aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc 6060
caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga 6120
aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag 6180
atcctcgccg tcgggcatcc gcgccttgag cctggcgaac agttcggctg gcgcgagccc 6240
ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc 6300
tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg 6360
cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga 6420
caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac 6480
aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc 6540
ctcgtcttgg agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg 6600
cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca 6660
gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg 6720
ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatcc actagattat tgaagcattt 6780
atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 6840
taggggttcc gcgcacattt ccccgaaaag tgccacctgc atcgatggcc cccgatggta 6900
gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct 6960
cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt 7020
aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg 7080
gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat 7140
ggcctttttg cgtggccagt gccaagcttg c 7171
<210> 2
<211> 7677
<212> DNA
＜213〉artificial sequence
<400> 2
agattgcagc attacacgtc ttgagcgatt gtgtaggctg gagctgcttc gaagttccta 60
tactttctag agaataggaa cttcggaata ggaacttcat ttactgcatc ctgtacttcg 120
caatgttgac ctcaaatatc ggaagtacac cgacgtattt cccggtgatc accgcacagg 180
aacttcgtgc gcataacgac aagctcttta gccctgagtt catcaaaaac atgctaaagt 240
atctctggga tcgcgacagc ggtgctggac tcagagctgc atcgggtttc cgcaacgtca 300
tgctcaaatc gggtcgccac attgatattc aacgcctcaa tgaaaaacag ctctttgttg 360
gactcaagcg cctgcttggt ctcctcggtc accagattct tgaatttgat cgtctctctg 420
gtgatgatgc ccatgttgat actaacgagg atgtacttga tctcattgcc tacaacgtct 480
cagacgtggt gggcaccaga ctgctcgctg aggacccggt gtactccggc tctttcgatc 540
tgcgggcagg tctactgagc acctacccag agactgtttt tgatcatgat ggtactttcc 600
gtcagccatc cacgcagatg cgtaaagatc gcctaacgat taatacctca tcagctcagt 660
tcgcagcgcg tattttggcg ccatatcgcc cactccgcga tgtccctgat gcgattggcg 720
acatgccggt ggtgtcttac ttgtacccgg atgcagcagt cgccgaagca acaggtcaaa 780
aacaagtcaa cgtgcttgat gagtcaaaga agttcttcta tgacaacatc accgacccgg 840
aagcacgtgc tgcctttgat gaggtctttg ctttttacgc tgatattgag ggtcgcaact 900
tcaacagtca caatgaggtt attgataccc agattaacca attacgtgct tatctcaacc 960
aggttgtcgc attcgatgca gctgggtatg cgctctatga tgtacgtaca cgttttgagc 1020
agatcttccc caaggatcgc agctacatca acgatgctac ggatatgacc cctcgcgcag 1080
tatcgagctt tgacgatctg gttgcactct gtgatgatat tcgcggtgta cttgatcgag 1140
gtttagagat ctcatctccg aatcatcatg agatggtgga tgctatgcgc aagcagctgc 1200
actatattca ggcattttac cgtgcctggg gacccattca acgccgcttc aatgacgctg 1260
acccagcggt gacccatccg catctcacag tgatctaccc accgctcacc cctgcatccg 1320
cagagaaatt caacaagatc acctcagtcg ctgctgtgag caagcgcacg cgttcgagaa 1380
atcataaaaa atttatttgc tttgtgagcg gataacaatt ataatagatt caattgtgag 1440
cggataacaa tttcacacag aattcattaa agaggagaaa ttaactatga gaggatcgca 1500
tcaccatcac catcacggat ccgcatgcga gctcggtacc ccgggtcgac ctgcagccaa 1560
gcttaattag ctgagcttgg actcctgttg atagatccag taatgacctc agaactccat 1620
ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc 1680
tagcttggcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata 1740
taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt 1800
tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt 1860
aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa 1920
tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt 1980
tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga 2040
ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg 2100
tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa 2160
tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc 2220
ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc 2280
gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt 2340
acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg 2400
cccttaaacg cctggggtaa tgactctcta gcttgaggca tcaaataaaa cgaaaggctc 2460
agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta 2520
ggacaaatcc gccctctaga gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg 2580
acacatgcag ctcccgtgca cgtgctggag aatattcggc aaggtctgaa ccgtcccagc 2640
catcgccatg aaagggttag gggctgtatg agcctgtttg ttgctggggt atttgcacaa 2700
tacggtcccc tcgcccctct ggggagaggg ttagggtgag gggaaaagcg ccccccctgc 2760
cgcagcctgc tccggtcgga cctggcaact atagctactc acagccaggt tgattataat 2820
aaccgtttat ctgttcgtac tgtttactaa aacgacgaat cgcctgattt tcaggcacaa 2880
caagcatcaa caataaggat taaagctatg ggttttcttt ccggtaagcg cattctggta 2940
accggtgttg ccagcaaact atccatcgcc tacggtatcg ctcaggcgat gcaccgcgaa 3000
ggagctgaac tggcattcac ctaccagaac gacaaactga aaggccgcgt agaagaattt 3060
gccgctcaat tgggttctga catcgttctg cagtgcgatg ttgcagaaga tgccagcatc 3120
gacaccatgt tcgctgaact ggggaaagtt tggccgaaat ttgacggttt cgtacactct 3180
attggttttg cacctggcga tcagctggat ggtgactatg ttaacgccgt tacccgtgaa 3240
ggcttcaaaa ttgcccacga catcagctcc tacagcttcg ttgcaatggc aaaagcttgc 3300
cgctccatgc tgaatccggg ttctgccctg ctgacccttt cctaccttgg cgctgagcgc 3360
gctatcccga actacaacgt tatgggtctg gcaaaagcgt ctctggaagc gaacgtgcgc 3420
tatatggcga acgcgatggg tccggaaggt gtgcgtgtta acgccatctc tgctggtccg 3480
atccgtactc tggcggcctc cggtatcaaa gacttccgca aaatgctggc tcattgcgaa 3540
gccgttaccc cgattcgccg taccgttact attgaagatg tgggtaactc tgcggcattc 3600
ctgtgctccg atctctctgc cggtatctcc ggtgaagtgg tccacgttga cggcggtttc 3660
agcattgctg caatgaacga actcgaactg aaataaatgc ataattccct gtacttcgca 3720
atgttgacct caaatatcgg aagtacaccg acgtatttcc cggtgatcac cgcacaggaa 3780
cttcgtgcgc ataacgacaa gctctttagc cctgagttca tcaaaaacat gctaaagtat 3840
ctctgggatc gcgacagcgg tgctggactc agagctgcat cgggtttccg caacgtcatg 3900
ctcaaatcgg gtcgccacat tgatattcaa cgcctcaatg aaaaacagct ctttgttgga 3960
ctcaagcgcc tgcttggtct cctcggtcac cagattcttg aatttgatcg tctctctggt 4020
gatgatgccc atgttgatac taacgaggat gtacttgatc tcattgccta caacgtctca 4080
gacgtggtgg gcaccagact gctcgctgag gacccggtgt actccggctc tttcgatctg 4140
cgggcaggtc tactgagcac ctacccagag actgtttttg atcatgatgg tactttccgt 4200
cagccatcca cgcagatgcg taaagatcgc ctaacgatta atacctcatc agctcagttc 4260
gcagcgcgta ttttggcgcc atatcgccca ctccgcgatg tccctgatgc gattggcgac 4320
atgccggtgg tgtcttactt gtacccggat gcagcagtcg ccgaagcaac aggtcaaaaa 4380
caagtcaacg tgcttgatga gtcaaagaag ttcttctatg acaacatcac cgacccggaa 4440
gcacgtgctg cctttgatga ggtctttgct ttttacgctg atattgaggg tcgcaacttc 4500
aacagtcaca atgaggttat tgatacccag attaaccaat tacgtgctta tctcaaccag 4560
gttgtcgcat tcgatgcagc tgggtatgcg ctctatgatg tacgtacacg ttttgagcag 4620
atcttcccca aggatcgcag ctacatcaac gatgctacgg atatgacccc tcgcgcagta 4680
tcgagctttg acgatctggt tgcactctgt gatgatattc gcggtgtact tgatcgaggt 4740
ttagagatct catctccgaa tcatcatgag atggtggatg ctatgcgcaa gcagctgcac 4800
tatattcagg cattttaccg tgcctgggga cccattcaac gccgcttcaa tgacgctgac 4860
ccagcggtga cccatccgca tctcacagtg atctacccac cgctcacccc tgcatccgca 4920
gagaaattca acaagatcac ctcagtcgct gctgtgagca agcgcccgcg gtggcgatga 4980
cgcatcctca cgataatatc cgggtaggcg caatcacttt cgtctctact ccgttacaaa 5040
gcgaggctgg gtatttcccg gcctttctgt tatccgaaat ccactgaaag cacagcggct 5100
ggctgaggag ataaataata aacgaggggc tgtatgcaca aagcatcttc tgttgagtta 5160
agaacgagta tcgagatggc acatagcctt gctcaaattg gaatcaggtt tgtgccaata 5220
ccagtagaaa cagacgaaga agctagcata aggcctcgca atggcttgca aggccacaca 5280
tgtattgaga tgttaataaa atgtagactt gtaattttga tataaatggt agagaaaatc 5340
tttccccaaa ataaaaacga acgtcaatga aatcaaacgg ttgaataaag ttgattttgg 5400
ctaatacaaa gacaagaaaa taatatttat gattaaatat cagcgagttg aatacataat 5460
ttttatatac tgctgcgcca tatgggctgg actgaagccg cagacctgat tgttaaaggt 5520
atggaaggcg caatcaacgc caagaccgta acctatgact tcgaacgtct gatggaaggc 5580
gctaaactgc tgaaatgcag cgagtttggt gacgcgatca tcaaaaatat gtaattacca 5640
catgtgttaa atattataac gggcgtataa cacgcccgtt gttttatgat gatgtaaaat 5700
cttccccaaa actttcccca aaacccttcc ccaaaactgg ctattttcta tgctgttttg 5760
atatctacga taatccagtc tttaccacga tcatcattgt atcggtcggt cattccatgg 5820
ctaattccca tgtcagccgt ctcgagttct gcgaagtgat cttccgtcac aggtaggcgc 5880
gccgaagttc ctatactttc tagagaatag gaacttcgga ataggaacta aggaggatat 5940
tcatatggac catggctaat tcccatgtca gccgttaagt gttcctgtgt cactcaaaat 6000
tgctttgaga ggctctaagg gcttctcagt gcgttacatc cctggcttgt tgtccacaac 6060
cgttaaacct taaaagcttt aaaagcctta tatattcttt tttttcttat aaaacttaaa 6120
accttagagg ctatttaagt tgctgattta tattaatttt attgttcaaa catgagagct 6180
tagtacgtga aacatgagag cttagtacgt tagccatgag agcttagtac gttagccatg 6240
agggtttagt tcgttaaaca tgagagctta gtacgttaaa catgagagct tagtacgtga 6300
aacatgagag cttagtacgt actatcaaca ggttgaactg ctgatcttca gatcctctac 6360
gccggacgca tcgtggccgg atcttgcggc cgcaaaaatt aaaaatgaag ttttgacggt 6420
atcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc 6480
tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca 6540
agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc 6600
agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag 6660
caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatccgcgc cttgagcctg 6720
gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca 6780
agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat 6840
gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact 6900
ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc 6960
agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc 7020
gtggccagcc acgatagccg cgctgcctcg tcttggagtt cattcagggc accggacagg 7080
tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca 7140
gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc 7200
ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct 7260
tgatccacta gattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 7320
tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 7380
acctgcatcg atggcccccg atggtagtgt ggggtctccc catgcgagag tagggaactg 7440
ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt 7500
gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg 7560
cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa 7620
ttaagcagaa ggccatcctg acggatggcc tttttgcgtg gccagtgcca agcttgc 7677
<210> 3
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 3
cttgcatgcc tgcaggtcga ct 22
<210> 4
<211> 29
<212> DNA
＜213〉artificial sequence
<400> 4
gaactcgaga cggctgacat gggaattag 29
<210> 5
<211> 29
<212> DNA
＜213〉artificial sequence
<400> 5
cgcctgcagt aaatgaagtt cctattccg 29
<210> 6
<211> 31
<212> DNA
＜213〉artificial sequence
<400> 6
gtcctcgagt tctgcgaagt gatcttccgt c 31
<210> 7
<211> 32
<212> DNA
＜213〉artificial sequence
<400> 7
cttatgcatc ctgtacttcg caatgttgac ct 32
<210> 8
<211> 40
<212> DNA
＜213〉artificial sequence
<400> 8
atactgcaga ctacgcgtgc gcttgctcac agcagcgact 40
<210> 9
<211> 32
<212> DNA
＜213〉artificial sequence
<400> 9
cttgaattcc ctgtacttcg caatgttgac ct 32
<210> 10
<211> 40
<212> DNA
＜213〉artificial sequence
<400> 10
atacaattgg gtccgcgggc gcttgctcac agcagcgact 40
<210> 11
<211> 29
<212> DNA
＜213〉artificial sequence
<400> 11
tcacaattgc ccgccctgcc actcatcgc 29
<210> 12
<211> 38
<212> DNA
＜213〉artificial sequence
<400> 12
actgaattcg cagtgcaccg cctacctgtg acggaaga 38
<210> 13
<211> 29
<212> DNA
＜213〉artificial sequence
<400> 13
ttaccgcggt ggcgatgacg catcctcac 29
<210> 14
<211> 29
<212> DNA
＜213〉artificial sequence
<400> 14
gaactcgaga cggctgacat gggaattag 29
<210> 15
<211> 26
<212> DNA
＜213〉artificial sequence
<400> 15
ccggtgcacg tgctggagaa tattcg 26
<210> 16
<211> 30
<212> DNA
＜213〉artificial sequence
<400> 16
gcgatgcatt tatttcagtt cgagttcgtt 30

Claims (5)

1. expression plasmid that is used for chromosomal integration, evolution, the multiple clone site, terminator, the resistance screening gene β that is used for the induced chromosome evolutionary process, phage integration site, and nucleotide sequence same clip A1 and the A2 of 2 allos being connected with resistance screening gene β both sides of promotor that contain 2 FRT sites, resistance screening gene a, replicon, promotor and back thereof, resistance screening gene a and resistance screening gene β are different resistant genes;
In described expression plasmid, the clooating sequence of each element is: the side in two FRT sites contain successively in the direction of the clock Segment A 1, promotor and back thereof multiple clone site, terminator, be used for resistance screening gene β, Segment A 2, the phage integration site of induced chromosome evolutionary process, the opposite side in two FRT sites contains resistance screening gene a and replicon;
Described resistance screening gene β for the induced chromosome evolutionary process is any of triclosan resistant gene, chloramphenicol resistance gene, ampicillin resistance gene, gentamicin resistant gene, trimethoprim resistant gene, spectinomycin resistance gene, streptomycin resistance gene, tetracycline resistance gene, lactobacillin resistant gene;
Described replicon is colibacillary condition replicon;
Described A1, A2 fragment are the allos fragment that comes from the other biological except intestinal bacteria, and the homology in intestinal bacteria is low and do not express, and size is 800～1200kbp.
2. expression plasmid according to claim 1, is characterized in that, described resistance screening gene a is kalamycin resistance gene.
3. expression plasmid according to claim 1, is characterized in that, described phage integration site is AttP HK, AttP P21, AttP λ, AttP φ 80With AttP P22In any.
4. expression plasmid according to claim 1, is characterized in that, described promotor is P T5 , P T7 , P tac , P trc , P lac, P BAD , P Ltet, P LlacEtc. any in promotor.
5. according to claim 1～4 described expression plasmids of any one, is characterized in that, the Host Strains that described expression plasmid transforms be intestinal bacteria ( Escherichia coli).
CN 201210060042 2012-03-08 2012-03-08 Expression plasmid for integrating and evolving chromosome Expired - Fee Related CN102559729B (en)

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GB2509159B (en) * 2012-12-21 2017-07-26 Oxoid Ltd Triclosan derivatives and uses thereof
CN104789586B (en) * 2015-04-23 2018-01-02 浙江大学 Genome of E.coli integration vector, genetic engineering bacterium and the application in xylitol is produced
CN106480016A (en) * 2016-09-29 2017-03-08 南京金斯瑞生物科技有限公司 A kind of method of extracting low-copy plasmid
CN112831453B (en) * 2019-11-25 2022-09-27 江南大学 Escherichia coli having amino acid oxidase incorporated therein
CN114891794B (en) * 2022-06-16 2023-07-21 中山大学 Promoter for regulating expression of tomato epicarp and application thereof

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