CN109055413A - A kind of shuttle vector and its construction method and application - Google Patents
A kind of shuttle vector and its construction method and application Download PDFInfo
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- CN109055413A CN109055413A CN201810853597.2A CN201810853597A CN109055413A CN 109055413 A CN109055413 A CN 109055413A CN 201810853597 A CN201810853597 A CN 201810853597A CN 109055413 A CN109055413 A CN 109055413A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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Abstract
The invention belongs to plamid vector construction field, a kind of shuttle vector and its construction method and application are provided.The shuttle vector be connect by Aeromonas hydrophila plasmid pBK760 with carrier pMD20 it is built-up.Shuttle vector provided by the invention can replicate in Escherichia coli and Aeromonas hydrophila.Genetic manipulation system is still immature in Aeromonas hydrophila, step of converting is cumbersome, and its transformation efficiency is relatively low, and gene cloning operation is very convenient in Escherichia coli.Exogenous gene cloning and construction of recombinant plasmid are completed in Escherichia coli using the shuttle vector, convenient for carrying out related gene functional study and genetic breeding work.
Description
Technical field
The invention belongs to plamid vector construction technical fields, more particularly, to a kind of shuttle vector and its building
Methods and applications.
Background technique
Aeromonas hydrophila is distributed widely in the various water bodys of nature, is the primary pathogenic bacteria of a variety of aquatic animals,
It is that typical people-beast-fish is total to illness pathogen, the dissociant of Aeromonas hydrophila is more, most bacterial strains for conditioned pathogen
Sensitive to orfloxacin, ceftriaxone, Aeromonas hydrophila shows multiple resistance, and one of main cause is the plasmid containing there are many,
A kind of this laboratory isolated plasmid from Aeromonas hydrophila, is named as plasmid pBK760, which has a variety of sites
And resistance, there are good vector properties.
PMD20 is a kind of special carrier of high-efficient cloning PCR product.The carrier is based on pUC19 carrier, at its more grams
Tri- kinds of Spe I, Nde I, EcoR V restriction enzyme sites are increased between Xba I and the BamH I site of Long Weidianchu, through EcoR V
3 ' end additions " T " after digestion again in two sides are prepared.Because having when most of Taq DNA polymerase carries out PCR reaction
In the characteristic of 3 ' end additions one " A " of PCR product, it is possible to greatly improve connection, the cloning efficiency of PCR product.
Although plasmid pBK760 and pMD20 can come separately as carrier using if its advantage can mutually be tied
It closes, so that it may play their own advantage, advantage of both the present invention combination plasmid pBK760 and pMD20 utilizes the modern times
Technology constructs a kind of completely new shuttle vector (pBKPU01), convenient for carrying out related gene functional study and genetic breeding
Work.
Summary of the invention
The object of the present invention is to provide a kind of completely new shuttle vectors and its construction method and application.
Specifically, the first aspect of the present invention provides a kind of shuttle vector, and the shuttle vector is by thermophilic aqueous vapor list
Born of the same parents' bacteria plasmid pBK760 connect built-up with carrier pMD20；The Aeromonas hydrophila plasmid pBK760 includes setting gradually
LacZ gene, replication origin and ampicillin resistance gene.
Specifically, the lacZ gene is located at 347~676 of plasmid pBK760, and the replication origin is located at plasmid
972~1560 of pBK760, the ampicillin resistance gene are located at 1833~2693 of plasmid pBK760.
Most specifically, the Aeromonas hydrophila plasmid pBK760 has nucleotide sequence (matter shown in SEQ ID NO:1
Grain map is as shown in Figure 3).
Further, the shuttle vector further include positioned at 3355~3561 promoter site, be located at 3726~
3812 signal DNA encoding peptide, positioned at 1592~2363 kalamycin resistance gene, positioned at 2531~2735 it is rich come it is mould
Plain resistant gene.
Carrier pMD20 can be commercially available.
Most specifically, the nucleotide sequence of the shuttle vector is as shown in SEQ ID NO:2.
The second aspect of the present invention provides a kind of construction method of shuttle vector, comprising the following steps:
(1) using Aeromonas hydrophila plasmid pBK760 as template, PCR is carried out by primer P1 and P2, is linearized
PBK760 plasmid recycles linearisation pBK760 plasmid after purification；
Shown in the nucleotide sequence of P1 such as SEQ ID NO:3 (5 '-ATCGTCGACCCTCTAGATGCAG-3 '), the core of P2
Shown in nucleotide sequence such as SEQ ID NO:4 (5 '-CTGACTCTAGCAGGTCGAGGAT-3 ')；
(2) with T4 ligase by after purification and recovery linearisation pBK760 plasmid and carrier pMD20 be attached；
(3) connection product is converted to competent escherichia coli cell (such as E.coli DH5 α competent cell), picking
Transformant, extraction obtain the shuttle vector.
The third aspect of the present invention provides the application of above-mentioned shuttle vector.
Specifically, the present invention provides the shuttle vector in screening Aeromonas hydrophila label resistant gene structure
Using.
Label resistant gene screening of the present invention is resistant gene will to be marked to be connected into the shuttle vector, then again
Obtained carrier is converted into competent escherichia coli cell, positive transformant is screened, positive transformant is finally transferred to thermophilic aqueous vapor
Monad obtains the Aeromonas hydrophila with label resistant gene.
Specifically, the application the present invention also provides the shuttle vector in Aeromonas hydrophila gene knockout.
Gene knockout of the present invention is that the homologous sequence of resistant gene, gene to be knocked out will be marked to be connected into the shuttle matter
Grain carrier, then converts competent escherichia coli cell for obtained carrier again, positive transformant is screened, finally by positive transformants
Son is transferred to Aeromonas hydrophila, obtains the Aeromonas hydrophila for knocking out target gene.
The beneficial effects of the present invention are:
Shuttle vector provided by the invention can replicate in Escherichia coli and Aeromonas hydrophila.Aeromonas hydrophila
Middle genetic manipulation system is still immature, step of converting is cumbersome, and its transformation efficiency is relatively low, and gene gram in Escherichia coli
Grand operation is very convenient.Exogenous gene cloning and construction of recombinant plasmid are completed in Escherichia coli using the shuttle vector,
Convenient for carrying out related gene functional study and genetic breeding work, conversion ratio is up to 7 × 105-8×105CFU/μg DNA。
Other features and advantages of the present invention will then part of the detailed description can be specified.
Detailed description of the invention
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its
Its purpose, feature and advantage will be apparent.
Fig. 1 is that shuttle vector pBKPU01 constructs schematic diagram.
Fig. 2 is shuttle vector pBKPU01 map.
Fig. 3 is plasmid pBK760 map.
Fig. 4 is gel electrophoresis figure of the plasmid pBK760 after PCR is linearized.
Electrophoretogram is identified in the single, double digestion of shuttle vector pBKPU01 that Fig. 5 is extracted after being Transformed E .coli DH5 α.
Specific embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe preferred implementations of the invention
Mode, however, it is to be appreciated that may be realized in various forms the present invention without that should be limited by the embodiments set forth herein.
The person that is not specified actual conditions in embodiment, all carries out according to conventional conditions or manufacturer's recommended conditions.Examination used
Production firm person is not specified in agent or instrument, is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
The present embodiment is used to illustrate the building of shuttle vector pBKPU01, and building schematic diagram is as shown in Figure 1.
1, the extraction of plasmid pBK760
Picking Aeromonas hydrophila single colonie is inoculated in LB liquid medium of the 5mL containing 50 μ g/mL kanamycins, 37
DEG C culture 12h.The a small amount of extraction agent boxes of bacterium solution Plasmid DNA (the raw work in Shanghai) extract plasmid pBK760, and -20 DEG C of storages are stand-by.
2, plasmid pBK760 is linearized
Using plasmid pBK760 as template (sequence is as shown in SEQ ID NO:1), carried out by setting primer (P1 and P2)
PCR linearizes cyclic plasmid pBK760, and the specific reaction condition of PCR is shown in Table 1, P1 and P2 sequence is as follows:
P1:5 '-ATCGTCGACCCTCTAGATGCAG-3 ' (SEQ ID NO:3)
P2:5 '-CTGACTCTAGCAGGTCGAGGAT-3 ' (SEQ ID NO:4)
Table 1:pBK760 linearizes PCR and reacts actual conditions
| Taq DNA polymerase buffer liquid | 5μL |
| dNTPs | 5μL |
| Primer P1 | 2.5μL |
| Primer P2 | 2.5μL |
| Template pBK760 | 2μL |
| Taq archaeal dna polymerase | 0.5μL |
| ddH2O | 32.5μL |
| All | 50μL |
Response procedures are as follows: 94 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 4.5min, 32 circulations；72 DEG C of extension 10min；4
DEG C save.
Product after PCR amplification is by the identification of 1% agarose gel electrophoresis, as shown in Figure 4.Electrophoresis result is shown in theory
There is single specific band in value size position, with Plasmid DNA segment QIAquick Gel Extraction Kit (purchased from the raw work in Shanghai) to its into
Row purification and recovery.
3, the connection of plasmid pBK760 and carrier pMD20
Linearization plasmid pBK760 in step 2 and carrier pMD20 (being purchased from TaKaRa company) are attached, reaction system
As shown in table 2.
Table 2: plasmid pBK760 and carrier pMD20 linked system
| T4DNA ligase buffer solution | 1μL |
| Linearization plasmid pBK760 | 2μL |
| PMD20 carrier | 6μL |
| T4DNA ligase | 1μL |
| All | 10μL |
Operating procedure is as follows: 16 DEG C of connections overnight, take 2.5 μ L connection products to convert 50 μ L E.coli DH5 α competence thin
Born of the same parents take LB plate of the 100 μ L bacterium solutions coating containing 100 μ g/mL ampicillins to carry out blue hickie screening.Picking positive transformants
Son, is transferred to the LB liquid medium that 5mL contains 100 μ g/mL ampicillins, and 37 DEG C of culture 12h extract examination with Plasmid DNA in a small amount
Agent box (purchased from the raw work in Shanghai) extracts plasmid, obtains shuttle vector pBKPU01.It is carried out by Hind III and EcoR I
Single, double digestion verification (as shown in Figure 5) carries out sequencing analysis (Hua Da gene) after determining recombinant plasmid.The sequencing results are such as
It is as shown in Figure 2 to draw Vector map by SEQ ID NO:2.
Embodiment 2
The present embodiment is used to illustrate the application of shuttle vector pBKPU01
1, shuttle vector pBKPU01 converts Aeromonas hydrophila
A, the preparation of Aeromonas hydrophila competence
1) the fresh Aeromonas hydrophila single colonie of picking is inoculated in 5mL LB liquid tube, and 37 DEG C, 200r/min training
It supports overnight.
2) bacterium solution is accessed in LB liquid medium of the 50mL containing 0.5mol/L by 5% (v/v) inoculum concentration, and 37 DEG C, 200r/
It is 2 or so that min, which is cultivated to OD600, takes 30min, 5000r/min, 4 DEG C of centrifugation 6min of whole bacterium solution ice baths, collects thallus.
3) with the Solution A of 10mL pre-cooling, (0.5mol/L sorbierite, the mannitol of 0.5mol/L, 10% glycerol are molten
In deionized water) thallus is resuspended, 5000r/min, 4 DEG C of centrifugation 8min go supernatant (eliminating as far as possible), so rinsing 3 times.
4) Solution B (0.5mol/L trehalose, 0.5mol/L sorbierite, the 0.5mol/L sweet dew being pre-chilled with 30mL
Alcohol, 10% glycerol, is dissolved in deionized water) washing thalline is primary, and 8000r/min, 4 DEG C of centrifugation 5min go supernatant (to remove as far as possible
To the greatest extent).
5) with 500 μ L Solution B suspension thallines, as Aeromonas hydrophila electricity turns competent cell, is distributed into 50 μ
The every pipe of L, -80 DEG C of preservations, for use.
B, transformed competence colibacillus cell
1) electrotransformation: the 50 thermophilic aqueous vapors of μ L of shuttle vector pBKPU01 and step A preparation prepared by 2 μ L embodiments 1
Monad electricity turns competent cell and softly mixes, ice bath 30min, mixture is transferred in the 1mm electric shock cup of pre-cooling, 2000V electricity
The resuscitation fluid for being once rapidly added 1mL pre-cooling later is hit, 37 DEG C, 200r/min cultivates 3h.
2) 100 μ L bacterium solutions are taken to be coated on the LB agar medium containing 50 μ g/mL kanamycins, 37 DEG C of culture 12h.
3) picking positive transformant, be transferred to 5mL contain 50 μ g/mL kanamycins LB liquid medium, 37 DEG C of culture 12h,
Plasmid is extracted with a small amount of extraction agent boxes of Plasmid DNA (purchased from the raw work in Shanghai), double digestion is carried out by Hind III and EcoR I
It verifies (such as Fig. 5), as a result proves really to be plasmid pBKPU01.
Above embodiments illustrate that shuttle vector pBKPU01 of the present invention is constructed successfully, which can be in thermophilic aqueous vapor list
Expression is replicated in born of the same parents bacterium.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill
Many modifications and changes are obvious for the those of ordinary skill in art field.
Sequence table
<110>Jiangsu medical profession institute
<120>a kind of shuttle vector and its construction method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3817
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agaacctaaa aagaacgaat ttgaactaac tcataaccga gaggtaaaaa aagaacgaag 60
tcgagatcag ggaatgagtt tataaaataa aaaaagcacc tgaaaaggtg tctttttttg 120
atggttttga acttgttctt tcttatcttg atacatatag aaataacgtc atttttattt 180
tagttgctga aaggtgcgtt gaagtgttgg tatgtatgtg ttttaaagta ttgaaaaccc 240
ttaaaattgg ttgcacagaa aaaccccatc tgttaaagtt ataagtgact aaacaaataa 300
ctaaatagat gggggtttct tttaatatta tgtgtcctaa tagtagcatt tattcagatg 360
agaaaatcgc taatgttgat tactttgaac ttctgcatat tcttgaattt aaaaaggctg 420
aaaaatcaag ggttttagtg gacaagacaa aaagtggaaa agtgagacca tggagagaaa 480
aaagagtaaa agattgtgct gaaatattag agtataaaca aaatcgtgaa acaggcgaaa 540
gaaagttgta tcgagtgtgg ttttgtaaat ccaggctttg tccaatgtgc aactggagga 600
gagcaatgaa acatggcatt cagtcacaaa aggttgttgc tgaagttatt aaacaaaagc 660
caacagttcg ttggttgttt ctcacattaa cagttaaaaa tgtttatgat ggcgaagaat 720
taaataagag tttgtcagat atggctcaag gatttcgccg aatgatgcaa tataaaaaaa 780
ttaataaaaa tcttgttggt tttatgcgtg caacggaagt gacaataaat aataaagata 840
cagaaaacta cgtgaatcaa aaacaatgga ttcaattttg gaaaaaggca atgaaattag 900
attcttataa tcagcacatg catgtattgg tatgtgtgga accaacttat tttaagaata 960
actatgatcc aaatgtaaaa gttcaaatga ttcgaccgaa aaataaatat aaatcggata 1020
tacaatcggc aattgacgaa actgcaaaat atcctgtaaa ggatacggat tttatgaccg 1080
atgatgaaga aaagaatttg aaacgtttgt ctgatttgga ggaaggttta caccgtaaaa 1140
ggttaatctc ctatggtggt ttgttaaaag aaatacataa aaaattaaac cttgatgaca 1200
cagaagaagg cgatttgatt catacagatg atgacgaaaa agccgatgaa gatggatttt 1260
ctattattgc aatgtggaat tgggaacgga aaaattattt tattaaagag tagttcaaca 1320
aacgggccag tttgttgaag attagatgct ataattgtta ttaaaaggat tgaaggatgc 1380
ttaggaagac gagttattaa tagctgaata agaacggtgc tctccaaata ttcttattta 1440
gaaaagcaaa tctaaaatta tctgaaaagg gaatgagaat agtgaatgga ccaataataa 1500
tgactagaga agaaagaatg aagattgttc atgaaattaa ggaacgaata ttggataaat 1560
atggggatga tgttaaggct attggtgttt atggctctct tggtcgtcag actgatgggc 1620
aagcccaaac gttccacgat gcgatttgtg cccttatcgt agaagagctg tttgaatatg 1680
aatggacaac cggtgagtgg aaggtggaag tgaattttga tagcgaagag attctactag 1740
attatgcatc tcaggtggaa tcagattggc cgcttacaca tggtcaattt ttctctattt 1800
tgccgattta tgattcaggt ggatacttag agaaagtgta tcaaactgct aaatcggtag 1860
cctattcgga tattgagatg atgtgtgtca tgtcaacaga ggaagcagag ttcagccatg 1920
caggcaaatg gcgtaatatt cgtgtgcaag gaccgacaac atttctacca tccttgactg 1980
tacaggtagc aatggcaggt gccatgttga ttggtctgca tcatcgcatc tgttatacga 2040
cgagcgcttc ggtcttaact gaagcagtta agcaatcaga tcttccttca ggttatgacc 2100
atctgtgcca gttcgtaatg tctggtcaac tttccgactc tgagaaactt ctggaatcgc 2160
ataaagggtg tgcttaaatc gggccatttt gcgtaataag aaaaaggatt aattatgagc 2220
tgtcaaaacg cataccattt tgaacgatga cctctaataa ttgttaatca tgttggttac 2280
gtatttatta acttctccta gtattagtaa ttatcatggc tgtcatggcg cattaacgga 2340
tagagaattt ctggaatggg attcaggagt ggacagaacg acacggatat atagtggatg 2400
gaattgaatt aataataagg taatagattt acattagaaa atgaaagggg attttatgcg 2460
tgagaatgtt acagtctatc ccggcattgc cagtcgggga tattaaaaag agtataggtt 2520
tttattgcga taaactaggt ttcactttgg ttcaccatga agatggattc gcagttctaa 2580
tgtgtaatga ggttcggatt catctatggg aggcaagtga tgaaggctgg cgctctcgta 2640
gtaatgattc accggtttgt acaggtgcgg agtcgtttat tgctggtact gctagttgcc 2700
gcattgaagt agagggaatt gatgaattat atcaacatat taagcctttg ggcattttgc 2760
accccaatac atcattaaaa gatcagtggt gggatgaacg agactttgca gtaattgatc 2820
tttaaacgag cacgagagca aaacccccct ttgctgaggt ggcagagggc aggttttttt 2880
gttcagcaat cgggcgcgat tgctgaataa aagatacgag agacctctct tgtatctttt 2940
ttattttgag tggttttgtc cgttacacta gaaaaccgaa agacaataaa aattttattc 3000
ttgctgagtc tggctttcgg taagctagac aaaacggaca aaataaaaat tggcaagggt 3060
ttaaaggtgg agattttttg agtgatcttc tcaaaaaata ctacctgtcc cttgctgatt 3120
ccgacaacaa tttgattagc ttttttcaac aaataaaaag ctaaaatcta ttattaatct 3180
gtttcttttt tctcgtaaaa aaaagaaagg tcttaaaggt tttatggttt tggtcggcac 3240
tgaattcgag ctcagcatta ttgagtggat gattatattc cttttgatag gtggtatgtt 3300
ttcgcttgaa cttttaaata cagccattga acatacggtt gatttaataa ctgacaaaca 3360
tcaccctctt gctaaagcgg ccaaggacgc tgccgccggg gctgtttgcg tttttgccgt 3420
gatttcgtgt atcattggtt tacttatttt tttgccaaag ctgtaatggc tgaaaattct 3480
tacatttatt ttacattttt agaaatgggc gtgaaaaaaa gcgcgcgatt atgtaaaata 3540
taaagtgata gcggtaccag gagggctgga agaagcagac cgctaacaca gtacataaaa 3600
aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt 3660
actaccgcac tgctggcagg aggcgcaact caagcttttg cctcgagctc ggtacccggg 3720
gatcctctag agtcgacctg caggcatgca agctagcttc agcacaattc caagaaagac 3780
gcggtaccag gagggctgga agaagcagac acgattt 3817
<210> 2
<211> 6635
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 240
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
cttccagaga ttatcctcta gagtcgacct gcagatcctc tagagtcgac ctgcaggcat 420
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggt acgcgcggat 480
gcaagctagc ttcagcacaa ttccaagaaa aacacgattt agaacctaaa aagaacgaat 540
tataaaataa aaaaagcacc tgaaaaggtg tctttttttg atggttttga acttgttctt 600
ttgaactaac tcataaccga gaggtaaaaa aagaacgaag tcgagatcag ggaatgagtt 660
tcttatcttg atacatatag aaataacgtc atttttattt tagttgctga aaggtgcgtt 720
tttaatatta tgtgtcctaa tagtagcatt tattcagatg aaaaatcaag ggttttagtg 780
aaaccccatc tgttaaagtt ataagtgact aaacaaataa ctaaatagat gggggtttct 840
gaagtgttgg tatgtatgtg ttttaaagta ttgaaaaccc ttaaaattgg ttgcacagaa 900
gacaagacaa aaagtggaaa agtgagacca tggagagaaa agaaaatcgc taatgttgat 960
tactttgaac ttctgcatat tcttgaattt aaaaaggctg aaagagtaaa agattgtgct 1020
gaaatattag agtataaaca aaatcgtgaa acaggcgaaa gaaagttgta tcgagtgtgg 1080
ttttgtaaat ccaggctttg tccaatgtgc aactggagga gagcaatgaa acatggcatt 1140
atggctcaag gatttcgccg aatgatgcaa tataaaaaaa ttaataaaaa tcttgttggt 1200
ctcacattaa cagttaaaaa tgtttatgat ggcgaagaat taaataagag tttgtcagat 1260
cagtcacaaa aggttgttgc tgaagttatt aaacaaaagc caacagttcg ttggttgttt 1320
tttatgcgtg caacggaagt gacaataaat aataaagata attcttataa tcagcacatg 1380
catgtattgg tatgtgtgga accaacttat tttaagaata cagaaaacta cgtgaatcaa 1440
aaacaatgga ttcaattttg gaaaaaggca atgaaattag actatgatcc aaatgtaaaa 1500
gttcaaatga ttcgaccgaa aaataaatat aaatcggata tacaatcggc aattgacgaa 1560
ttgttaaaag aaatacataa aaaattaaac cttgatgaca cagaagaagg cgatttgatt 1620
aaacgtttgt ctgatttgga ggaaggttta caccgtaaaa ggttaatctc ctatggtggt 1680
actgcaaaat atcctgtaaa ggatacggat tttatgaccg atgatgaaga aaagaatttg 1740
catacagatg atgacgaaaa agccgatgaa gatggatttt ctattattgc aatgtggaat 1800
tgggaacgga aaaattattt tattaaagag tagttcaaca aacgggccag tttgttgaag 1860
attagatgct ataattgtta ttaaaaggat tgaaggatgc ttaggaagac gagttattaa 1920
tagctgaata agaacggtgc tctccaaata ttcttattta gaaaagcaaa tctaaaatta 1980
tctgaaaagg gaatgagaat agtgaatgga ccaataataa tgactagaga agaaagaatg 2040
atgtgtgtca tgtcaacaga ggaagcagag ttcagccatg aatggacaac cggtgagtgg 2100
attggtgttt atggctctct tggtcgtcag actgatgggc cctattcgga tattgagatg 2160
aagattgttc atgaaattaa ggaacgaata ttggataaat atggggatga tgttaaggct 2220
aaggtggaag tgaattttga tagcgaagag attctactag attatgcatc tcaggtggaa 2280
tcagattggc cgcttacaca tggtcaattt ttctctattt tgccgattta tgattcaggt 2340
ggatacttag agaaagtgta tcaaactgct aaatcggtag aagcccaaac gttccacgat 2400
gccatgttga ttggtctgca tcatcgcatc tgttatacga cgagcgcttc ggtcttaact 2460
cgtgtgcaag gaccgacaac atttctacca tccttgactg tacaggtagc aatggcaggt 2520
gcgatttgtg cccttatcgt agaagagctg tttgaatatg caggcaaatg gcgtaatatt 2580
gaagcagtta agcaatcaga tcttccttca ggttatgacc atctgtgcca gttcgtaatg 2640
tctggtcaac tttccgactc tgagaaactt ctggaatcgc tagagaattt ctggaatggg 2700
attcaggagt ggacagaacg acacggatat atagtggatg tgtcaaaacg cataccattt 2760
tgaacgatga cctctaataa ttgttaatca tgttggttac gtatttatta acttctccta 2820
gtattagtaa ttatcatggc tgtcatggcg cattaacgga ataaagggtg tgcttaaatc 2880
gggccatttt gcgtaataag aaaaaggatt aattatgagc gaattgaatt aataataagg 2940
ttcactttgg ttcaccatga agatggattc gcagttctaa tgtgtaatga ggttcggatt 3000
ccggcattgc cagtcgggga tattaaaaag agtataggtt tttattgcga taaactaggt 3060
taatagattt acattagaaa atgaaagggg attttatgcg tgagaatgtt acagtctatc 3120
catctatggg aggcaagtga tgaaggctgg cgctctcgta gtaatgattc accggtttgt 3180
acaggtgcgg agtcgtttat tgctggtact gctagttgcc gcattgaagt agagggaatt 3240
gatgaattat atcaacatat taagcctttg ggcattttgc accccaatac atcattaaaa 3300
cgttacacta gaaaaccgaa agacaataaa aattttattc ttgctgagtc tggctttcgg 3360
ttttttcaac aaataaaaag ctaaaatcta ttattaatct gttcagcaat cgggcgcgat 3420
tgctgaataa aagatacgag agacctctct tgtatctttt ttattttgag tggttttgtc 3480
gatcagtggt gggatgaacg agactttgca gtaattgatc ccgacaacaa tttgattagc 3540
taagctagac aaaacggaca aaataaaaat tggcaagggt ttaaaggtgg agattttttg 3600
agtgatcttc tcaaaaaata ctacctgtcc cttgctgatt tttaaacgag cacgagagca 3660
aaacccccct ttgctgaggt ggcagagggc aggttttttt gtttcttttt tctcgtaaaa 3720
aaaagaaagg tcttaaaggt tttatggttt tggtcggcac tgaattcgag ctcagcatta 3780
ttgagtggat gattatattc cttttgatag gtggtatgtt ttcgcttgaa cttttaaata 3840
tacttatttt tttgccaaag ctgtaatggc tgaaaattct tacatttatt ttacattttt 3900
ccaaggacgc tgccgccggg gctgtttgcg tttttgccgt gatttcgtgt atcattggtt 3960
cagccattga acatacggtt gatttaataa ctgacaaaca tcaccctctt gctaaagcgg 4020
agaaatgggc gtgaaaaaaa gcgcgcgatt atgtaaaata taaagtgata gcggtaccag 4080
gagggctgga agaagcagac cgctaacaca gtacataaaa aaggagacat gaacgatgaa 4140
catcaaaaag tttgcaaaac aagcaacagt attaaccttt actaccgcac tgctggcagg 4200
aggcgcaact caagcttttg cctcgagctc ggtacccggg tatcctctag agtcgacctg 4260
cagaatcgtc gaacggcagg cgtgcaaact tggcgtaatc atggtcatag ctgtttcctg 4320
tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 4380
aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 4440
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 4500
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 4560
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 4620
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 4680
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 4740
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 4800
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 4860
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 4920
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4980
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 5040
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 5100
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 5160
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 5220
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 5280
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 5340
aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 5400
ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 5460
acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 5520
ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 5580
gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 5640
taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 5700
tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 5760
gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 5820
cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 5880
aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 5940
cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 6000
ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 6060
gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 6120
tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 6180
gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 6240
tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 6300
cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 6360
agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatgg 6420
gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 6480
tgacattaac ctataaaaat aggcgtatca cgaggccctt tggtgtcacg ctcgtcgttt 6540
gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 6600
tgacattaac ctataaaaat aggcgtatca tcgtc 6635
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atcgtcgacc ctctagatgc ag 22
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ctgactctag caggtcgagg at 22
Claims (9)
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1401773A (en) * | 2001-08-20 | 2003-03-12 | 中国农业科学院植物保护研究所 | Bt gene with high toxicity to Lepidoptera and Coleoptera insects, expression vector and engineering bacteria |
| CN101781634A (en) * | 2009-12-04 | 2010-07-21 | 天津大学 | Recombinant zymomonas mobilis capable of producing ethanol by using xylose and fermentation method thereof |
| CN104031932A (en) * | 2014-06-27 | 2014-09-10 | 四川大学 | Zymomonas mobilis-escherichia coli shuttle vector pSUZM1, pSUZM2 and pSUZM3 and construction method thereof |
| CN104087610A (en) * | 2014-07-23 | 2014-10-08 | 中国科学院武汉病毒研究所 | Shuttle plasmid vector, as well as construction method and applications thereof |
| CN104212829A (en) * | 2014-06-30 | 2014-12-17 | 四川大学 | Series of Zymomonas mobilis expression vectors pSUZM, and construction method thereof |
| CN104313046A (en) * | 2014-10-15 | 2015-01-28 | 南京师范大学 | Microbial whole-cell luminescence reporter sensor for monitoring Hg2+ pollution, kit and application thereof |
| CN104962572A (en) * | 2015-06-29 | 2015-10-07 | 郑宗林 | Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein |
| CN107217025A (en) * | 2017-06-09 | 2017-09-29 | 盐城工学院 | A kind of bacillus subtilis JG 1 for producing endo-inulinase and its preparation method and application |
| CN107760705A (en) * | 2017-09-25 | 2018-03-06 | 浙江工业大学 | A kind of shuttle vector and its construction method and application |
-
2018
- 2018-07-30 CN CN201810853597.2A patent/CN109055413B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1401773A (en) * | 2001-08-20 | 2003-03-12 | 中国农业科学院植物保护研究所 | Bt gene with high toxicity to Lepidoptera and Coleoptera insects, expression vector and engineering bacteria |
| CN101781634A (en) * | 2009-12-04 | 2010-07-21 | 天津大学 | Recombinant zymomonas mobilis capable of producing ethanol by using xylose and fermentation method thereof |
| CN104031932A (en) * | 2014-06-27 | 2014-09-10 | 四川大学 | Zymomonas mobilis-escherichia coli shuttle vector pSUZM1, pSUZM2 and pSUZM3 and construction method thereof |
| CN104212829A (en) * | 2014-06-30 | 2014-12-17 | 四川大学 | Series of Zymomonas mobilis expression vectors pSUZM, and construction method thereof |
| CN104087610A (en) * | 2014-07-23 | 2014-10-08 | 中国科学院武汉病毒研究所 | Shuttle plasmid vector, as well as construction method and applications thereof |
| CN104313046A (en) * | 2014-10-15 | 2015-01-28 | 南京师范大学 | Microbial whole-cell luminescence reporter sensor for monitoring Hg2+ pollution, kit and application thereof |
| CN104962572A (en) * | 2015-06-29 | 2015-10-07 | 郑宗林 | Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein |
| CN107217025A (en) * | 2017-06-09 | 2017-09-29 | 盐城工学院 | A kind of bacillus subtilis JG 1 for producing endo-inulinase and its preparation method and application |
| CN107760705A (en) * | 2017-09-25 | 2018-03-06 | 浙江工业大学 | A kind of shuttle vector and its construction method and application |
Non-Patent Citations (1)
| Title |
|---|
| 邹少兰 等: "运动发酵单胞菌和大肠杆菌间穿梭质粒的构建", 《南开大学学报（自然科学版）》 * |
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