CN112359055A - Plasmid pMMB1 for enterobacter gene editing and construction method and application thereof - Google Patents

Abstract

The invention provides a plasmid pMMB1, the nucleotide sequence of which is shown in SEQ ID NO. 1. The invention also provides a construction method of the plasmid pMMB 1. The invention also provides application of the plasmid pMMB1 in gene editing. The plasmid provided by the invention has a conjugative transfer site oriT (RP4), and can be transferred into a receptor strain by modes of conjugative transfer, transformation and the like; has Multiple Cloning Sites (MCS) and can insert exogenous fragments conveniently and rapidly. Meanwhile, the plasmid can be transferred into an enterobacteriaceae receptor strain in various ways and used for gene editing of enterobacteriaceae bacteria.

Description

Plasmid pMMB1 for enterobacter gene editing and construction method and application thereof

Technical Field

The invention relates to a plasmid pMMB1 for enterobacter gene editing and a construction method and application thereof, belonging to the technical field of biology.

Background

In genetic engineering, plasmids are commonly used genetic vectors for carrying foreign genes and transferring them into host cells. Most of the common genetically engineered plasmids are derived from Escherichia coli. In order to achieve gene editing in a host cell, it is necessary to provide screening conditions outside to integrate the plasmid into a specific site on the host chromosome. This requires that the plasmid have the following characteristics: 1. has proper screening marks, and is convenient for screening to obtain recombinant transformants transferred into plasmids; 2. the plasmid is unable to replicate within the host cell, such that external selection pressure forces integration of the plasmid into the host chromosome. 3. The plasmid carries sequences homologous to the host gene and can therefore be integrated into a specific site by homologous recombination.

The origin of replication (oriV) of a genetically engineered plasmid is typically a deoxyribonucleotide sequence that depends on the replication elements of the host cell for plasmid replication. It can be divided into several groups according to its replication mechanism and host type. Since most of the genetically engineered plasmids are derived from Escherichia coli of Enterobacteriaceae, the replication origin thereof can be recognized in cells of various organisms of the Enterobacteriaceae family, so that the plasmids can replicate without integration into the chromosome of the host cell of the Enterobacteriaceae family, and thus gene editing of the host cell cannot be performed.

Disclosure of Invention

The technical problem is as follows: in order to solve the defects of the prior art, the invention aims to construct a genetic engineering plasmid pMMB1 which can be used for enterobacteriaceae gene editing and apply the plasmid to the work of carrying out gene editing on enterobacteriaceae hosts.

The technical scheme is as follows: the invention provides a plasmid pMMB1, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The invention provides a plasmid pMMB1, wherein the nucleotide sequence of the plasmid is a kanamycin resistance gene and a promoter region thereof from the 208-nd 1140-th deoxyribonucleotide at the 5' end; the 1192-position 3053 deoxyribonucleotide is a fructan sucrase gene sacB and a promoter region thereof; 3407-3516 shows that the deoxyribonucleotide is an IncP group plasmid conjugation transfer site; the 4497-4944 deoxyribonucleotide is a lambda pir protein-dependent replicon oriR 6K; 5287-5586-position deoxyribonucleotide is lacZ alpha gene for screening blue white spots and a promoter region thereof; the 5383-5417-site deoxyribonucleotide is a multiple cloning site for inserting an exogenous fragment.

The invention also provides a construction method of the plasmid pMMB1, which comprises the following steps:

(1) preparation of the cleavage product of the lambda pir protein-dependent replicon oriR 6K:

(1.1) carrying out PCR amplification on the lambda pir protein-dependent replicon oriR6K by using the plasmid pKNG101 as a template to obtain an amplification product; recovering the amplification product using a universal nucleic acid recovery kit to obtain a lambda pir protein-dependent replicon oriR 6K;

(1.2) treating the recovered amplification product with restriction endonuclease XhoI to obtain a lambda pir protein-dependent replicon oriR6K enzyme-cleaved product, and recovering the product by using a general-purpose nucleic acid recovery kit;

(2) preparation of plasmid backbone digestion product:

(2.1) PCR-amplifying sequences except the replicon by using an upstream primer DoriF and a downstream primer DoriR with the plasmid pK19mobsacB as a template to obtain an amplification product. Recovering the amplification product by using a universal nucleic acid recovery kit to obtain a plasmid skeleton;

(2.2) treating the recovered amplification product with restriction endonuclease XhoI to obtain a plasmid backbone enzyme digestion product, and recovering the enzyme digestion product by using a universal nucleic acid recovery kit;

(3) preparation of plasmid pMMB 1:

and connecting the lambda pir protein dependent replicon oriR6K enzyme digestion product with the plasmid skeleton enzyme digestion product to obtain the pMMB1 plasmid.

Wherein, in the step (1.1),

the amplification primers in the PCR amplification system are as follows:

the upstream primer R6 KF: 5-CCGCTCGAGTCTGAAGATCAGCAGTTCAACCTG-3;

the downstream primer R6 KR: 5-CCGCTCGAGAGCTTTGTCATCACCAATTTCCTT-3;

the PCR amplification system is 25 μ L, and comprises:

10 XDNA polymerase buffer 2.5. mu.L; 0.2mmol/L dATP; 0.2mmol/L dTTP; 0.2mmol/L dCTP; 0.2mmol/L dGTP; the upstream primer R6KF 0.3 is 0.3 mu mol/L; the downstream primer R6KR 0.3 is 0.3 mu mol/L; 0.1 mu g of template DNA; KOD DNA polymerase 0.5U, magnesium ion 1.5mmol/L, make up the remaining volume with sterilized double distilled water;

the reaction conditions for PCR amplification are as follows:

pre-denaturation at 98 ℃ for 5 min, after the pre-denaturation had ended the following procedure was carried out: denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 30 seconds, extension at 68 ℃ for 20 seconds, repeating for 30 cycles; after the circulation was completed, the temperature was 68 ℃ for 10 minutes.

Wherein, in the step (2.1),

the amplification primers in the PCR amplification system are as follows:

DoriF：5-CCGCTCGAGGAGCCTATGGAAAAACGCCAGCAACGCGGC-3；

DoriR：5-CCGCTCGAGTCTCAAGAAGATCCTTTGATCTTTTCTACG-3；

the PCR amplification system is 25 μ L, and comprises:

10 XDNA polymerase buffer 2.5. mu.L; 0.2mmol/L dATP; 0.2mmol/L dTTP; 0.2mmol/L dCTP; 0.2mmol/L dGTP; upstream primer DoriF 0.3 mu mol/L; downstream primer DoriR 0.3 mu mol/L; 0.1 mu g of template DNA; KOD DNA polymerase 0.5U, magnesium ion 1.5mmol/L, make up the remaining volume with sterilized double distilled water;

the reaction conditions for PCR amplification are as follows:

pre-denaturation at 98 ℃ for 5 min, after the pre-denaturation had ended the following procedure was carried out: denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 30 seconds, extension at 68 ℃ for 3 minutes, and 30 cycles were repeated. After the circulation was completed, the temperature was 68 ℃ for 10 minutes.

The invention also provides application of the plasmid pMMB1 in gene editing.

Has the advantages that: the plasmid provided by the invention has a conjugative transfer site oriT (RP4), and can be transferred into a receptor strain by modes of conjugative transfer, transformation and the like; has Multiple Cloning Sites (MCS) and can insert exogenous fragments conveniently and rapidly. Meanwhile, the plasmid can be transferred into an enterobacteriaceae receptor strain in various ways and used for gene editing of enterobacteriaceae bacteria.

Drawings

FIG. 1 is a physical map of plasmid pMMB 1;

FIG. 2 is a single-crossover detection electrophoretic analysis of the knockdown of the prodigiosin synthesis gene pigA of Serratia sp.ATCC 39006 by using pMMB1 plasmid in example 2 of the present invention;

FIG. 3 is a double crossover detection electrophoretic analysis of the knockdown of the prodigiosin synthesis gene pigA of Serratia sp.ATCC 39006 by using pMMB1 plasmid in example 2 of the present invention;

FIG. 4 is a photograph comparing colonies of the wild-type Serratia sp.ATCC 39006 strain and the pig A deletion mutant strain cultured on LB solid medium at 28 ℃ for 12 hours.

Detailed Description

The following examples are given to further describe the present invention in detail with reference to specific embodiments. The following examples are intended to illustrate the invention, but not to limit the scope of the invention.

The experimental procedures in the following examples are conventional, except for the specific illustrations. The raw materials and test reagents used in the examples were commercially available products except for those specifically mentioned.

In the following examples, the LB plate comprises: 10g/L tryptone; 10g/L of sodium chloride; 5g/L yeast extract; 20g/L agar powder. The LB liquid medium comprises: 10g/L tryptone; 10g/L of sodium chloride; 5g/L yeast extract. The M9 plate comprises: 12.8g/L of Na₂HPO₄·H₂O; KH of 3g/L₂PO₄(ii) a 0.5g/L NaCl; 1.0g/L NH₄Cl; 2mmol/L MgSO₄0.4g/L glucose, 0.1mmol/L CaCl₂20g/L agar powder. The LC liquid medium comprises: 10g/L tryptone; 5g/L (w/v) of yeast extract. The LCS plate comprises: 10g/L tryptone; 5g/L yeast extract; 20g/L of sucrose; 20g/L agar powder.

Example 1: construction of the plasmid pMMB1 for Enterobacter Gene editing described in the present invention

PCR amplification was performed using pNKG 101 plasmid as a template and the primer pair R6KF/R6KR to obtain the amplification product lambda pir protein-dependent replicon oriR 6K. And recovering the amplification product by using a universal nucleic acid recovery kit.

Wherein, the amplification primers R6KF and R6KR in the PCR amplification system are respectively:

R6KF：5-CCGCTCGAGTCTGAAGATCAGCAGTTCAACCTG-3

R6KR：5-CCGCTCGAGAGCTTTGTCATCACCAATTTCCTT-3

the PCR amplification system is 25 μ L, and comprises:

10 XDNA polymerase buffer 2.5. mu.L; magnesium ion 1.5 mmol/L; 0.2mmol/L dATP; 0.2mmol/L dTTP; 0.2mmol/L dCTP; 0.2mmol/L dGTP; the upstream primer R6KF 0.3 is 0.3 mu mol/L; the downstream primer R6KR 0.3 is 0.3 mu mol/L; 0.1 mu g of template DNA; KOD high fidelity DNA polymerase 0.5U; the remaining volume was replenished with sterile double distilled water.

The reaction conditions for PCR amplification are as follows:

pre-denaturation at 98 ℃ for 5 min, after the pre-denaturation had ended the following procedure was carried out: denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 30 seconds, and extension at 68 ℃ for 20 seconds, followed by 30 cycles. After the circulation was completed, the temperature was 68 ℃ for 10 minutes.

The recovered amplification product was treated with restriction endonuclease XhoI to obtain a lambda pir protein-dependent replicon oriR6K enzyme-cleaved product, which was recovered using a general-purpose nucleic acid recovery kit.

The DoriF/DoriR was PCR-amplified using the primers using plasmid pK19mobsacB as a template to obtain an amplification product. The amplification product is a sequence other than the pK19mobsacB plasmid replicon. And recovering the amplification product by using a universal nucleic acid recovery kit to obtain the plasmid skeleton.

Wherein, the amplification primers DoriF and DoriR in the PCR amplification system are respectively:

DoriF：5-CCGCTCGAGGAGCCTATGGAAAAACGCCAGCAACGCGGC-3

DoriR：5-CCGCTCGAGTCTCAAGAAGATCCTTTGATCTTTTCTACG-3

the PCR amplification system is 25 μ L, and comprises:

10 XDNA polymerase buffer 2.5. mu.L; magnesium ion 1.5 mmol/L; 0.2mmol/L dATP; 0.2mmol/L dTTP; 0.2mmol/L dCTP; 0.2mmol/L dGTP; upstream primer DoriF 0.3 mu mol/L; downstream primer DoriR 0.3 mu mol/L; 0.1 mu g of template DNA; KOD DNA polymerase 0.5U; the remaining volume was replenished with sterile double distilled water.

The reaction conditions for PCR amplification are as follows:

pre-denaturation at 98 ℃ for 5 min, after the pre-denaturation had ended the following procedure was carried out: denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 30 seconds, extension at 68 ℃ for 3 minutes, and 30 cycles were repeated. After the circulation was completed, the temperature was 68 ℃ for 10 minutes.

And (3) treating the recovered amplification product by using restriction endonuclease XhoI to obtain a plasmid backbone enzyme digestion product, and recovering the plasmid backbone enzyme digestion product by using a universal nucleic acid recovery kit.

And (3) connecting the cut product of the lambda pir protein-dependent replicon oriR6K with the cut product of the plasmid skeleton to obtain the pMMB1 plasmid (shown in the attached figure 1), and after a colony PCR is identified as a positive clone, extracting the plasmid and sequencing to determine the nucleotide sequence (shown in SEQ ID NO. 1).

Example 2: method for constructing gene knockout plasmid by using pMMB1 plasmid

In this example, a method of constructing a gene knockout vector pMMB1-DpigA for knocking out the pigA gene (Ser39006_020695) in Serratia sp.ATCC 39006 by using the pMMB1 plasmid in example 1 is described. The method comprises the following steps:

the pMMB1 plasmid is cut by restriction endonuclease PstI and restriction endonuclease BamHI, and the cut pMMB1 plasmid is recovered by a general nucleic acid purification and recovery kit;

performing PCR amplification on an upstream homology arm and a downstream homology arm of a pigA gene by using Serratia sp.ATCC 39006 genome DNA as a template to obtain upstream homology arm fragments and downstream homology arm fragments, and purifying and recovering the upstream homology arm fragments and the downstream homology arm fragments by using a universal nucleic acid purification and recovery kit;

wherein: the upstream primer D5F and the downstream primer D5R for amplifying the upstream homology arm are respectively:

D5F：5-TGCCTGCAGCTATCGGTTACTCACGCAACATGG-3

D5R：5-TGAGAATTCCTGACTATTTGACAGGTTAAAATCCAT-3

wherein, the upstream primer D3F and the downstream primer D3R for amplifying the downstream homology arm are respectively:

D3F：5-AGGGAATTCGGAACATCATCATGATTATTCAAC-3

D3R：5-GAAGGATCCATATTATCGAGCAACTCCATATGC-3

the PCR amplification system is 25 μ L, and comprises:

10 XDNA polymerase buffer 2.5. mu.L; magnesium ion 1.5 mmol/L; 0.2mmol/L dATP; 0.2mmol/L dTTP; 0.2mmol/L dCTP; 0.2mmol/L dGTP; upstream primer 0.3 mu mol/L; downstream primer 0.3 mu mol/L; 0.1 mu g of template DNA; KOD DNA polymerase 0.5U; the remaining volume was replenished with sterile double distilled water.

The reaction conditions for PCR amplification are as follows:

pre-denaturation at 98 ℃ for 5 min, after the pre-denaturation had ended the following procedure was carried out: denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 30 seconds, and extension at 68 ℃ for 30 seconds, followed by 30 cycles. After the circulation was completed, the temperature was 68 ℃ for 10 minutes.

Using restriction endonucleases PstI and EcoRI to enzyme-cut the upstream homologous arm fragment, purifying and recovering an enzyme-cut product, using restriction endonucleases EcoRI and BamHI to enzyme-cut the downstream homologous arm fragment, purifying and recovering an enzyme-cut product, and obtaining an enzyme-cut upstream homologous arm fragment and an enzyme-cut downstream homologous arm fragment;

connecting the enzyme-cut upstream homologous arm fragment and the enzyme-cut downstream homologous arm fragment with the enzyme-cut pMMB1 plasmid to obtain a connection product of a gene knockout vector pMMB 1-DpigA;

adding 20 mu L of the pMMB1-DpigA ligation product into 100 mu L of Escherichia coli S17-1 (lambda pir) competent cells prepared by a calcium chloride method, uniformly mixing, and standing in an ice bath for 30 minutes;

the mixture is thermally shocked in a water bath kettle at 42 ℃ for 90 seconds and then is ice-cooled for 5 minutes again;

adding 900 μ L LB liquid medium to the mixture, rejuvenating on a constant temperature shaker at 37 deg.C and 200rpm for 45 min, spreading on LB plate containing 50 μ g/mL kanamycin, and culturing overnight in 37 deg.C incubator;

selecting recombinant colonies on the culture medium, carrying out PCR amplification on the recombinant colonies by using detection primers UF and DR, detecting an amplification product by using agarose gel electrophoresis, and finally screening to obtain a positive colony containing a successfully-connected pMMB1-DpigA plasmid;

wherein: the sequences of the detection primers UF and DR are as follows:

UF：5-TAACCATGTTACTGGTAACTGGAA-3

DR：5-CACCACAAAGACAGCCATCATCAA-3

the template DNA of PCR is DNA extracted from somatic cells;

the PCR amplification system is 20 μ L, and comprises:

2 XTaq DNA polymerase buffer 10. mu.L; upstream primer 0.3 mu mol/L; downstream primer 0.3 mu mol/L; 0.1 mu g of template DNA; the remaining volume was replenished with sterile double distilled water.

The reaction conditions for PCR amplification are as follows:

pre-denaturation at 94 ℃ for 10 min, after the pre-denaturation was complete the following procedure was carried out: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds, followed by 30 cycles. After the circulation was completed, the temperature was 72 ℃ for 10 minutes.

The positive colonies containing successfully ligated pMMB1-DpigA plasmid were DNA sequenced to ensure the correctness of the pMMB1-DpigA plasmid sequence. The pMMB1-DpigA plasmid sequence is shown in SEQ ID NO. 2.

Example 3: method for gene knockout by using pMMB1-DpigA plasmid

This example describes a method for knocking out the pigA gene in Serratia sp.ATCC 39006 by using the pMMB1-DpigA plasmid in example 2. The pigA gene codes for a key enzyme in the process of prodigiosin synthesis, so that the Serratia sp.ATCC 39006 can not produce prodigiosin any more after the gene is knocked out. The method comprises the following steps:

inoculating Serratia sp.ATCC 39006 strain in LB liquid culture medium, and culturing at 30 deg.C and 200rpm in constant temperature shaking table overnight;

coli S17-1 (. lamda. pir) containing the above pMMB1-DpigA plasmid was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin. Culturing the liquid culture medium inoculated with the strains in a constant temperature shaking table at 37 ℃ and 220rpm overnight;

centrifuging the culture solution of Escherichia coli S17-1 (. lamda. pir) containing pMMB1-DpigA plasmid in a high speed centrifuge at 12000rpm for 30 seconds, discarding the supernatant, washing the precipitate twice with LB liquid medium to obtain Escherichia coli S17-1 (. lamda. pir) containing pMMB1-DpigA plasmid;

centrifuging the overnight-cultured Serratia sp.ATCC 39006 in a high-speed centrifuge at 12000rpm for 30 seconds to obtain Serratia sp.ATCC 39006 cells;

resuspending the Serratia sp.ATCC 39006 cell and the Escherichia coli S17-1 (. lamda. pir) cell containing pMMB1-DpigA plasmid in a fresh LB liquid medium, dropping the mixed cell solution on an LB plate, and culturing at 30 ℃ for 12 hours;

scraping the lawn from the LB plate and applying sterile 10mM MgSO₄The solution was washed 2 times to remove the attached nutrients. The washed cells were sterilized with 10mM MgSO₄The solution was resuspended, gradient diluted and plated on M9 plates containing 50. mu.g/mL kanamycin and incubated for 48 hours at 30 ℃ in an incubator;

selecting the recombinant colony on the M9 plate, carrying out PCR amplification on the recombinant colony by using detection primers UF and DR, detecting an amplification product by using agarose gel electrophoresis (shown in figure 2), and finally screening to obtain a positive colony integrating a pMMB1-DpigA plasmid into a genome, namely a pigA gene single-exchange strain;

wherein: the sequences of the detection primers UF and DR are as follows:

UF：5-TAACCATGTTACTGGTAACTGGAA-3

DR：5-CACCACAAAGACAGCCATCATCAA-3

the template DNA of PCR is DNA extracted from somatic cells;

the PCR amplification system is 20 μ L, and comprises:

2 XTaq DNA polymerase buffer 10. mu.L; upstream primer 0.3 mu mol/L; downstream primer 0.3 mu mol/L; 0.1 mu g of template DNA; the remaining volume was replenished with sterile double distilled water.

The reaction conditions for PCR amplification are as follows:

pre-denaturation at 94 ℃ for 10 min, after the pre-denaturation was complete the following procedure was carried out: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 1.5 minutes, and 30 cycles were repeated. After the circulation is finished, the temperature is 72 ℃ for 10 minutes

After the correct single-crossover strain of the pigA gene is picked, four-zone streaking is carried out on an LB flat plate containing 50 mu g/mL kanamycin, a single colony is separated, the single colony is inoculated in an LB liquid culture medium containing 50 mu g/mL kanamycin and is cultured in a constant-temperature shaking table at 30 ℃ and 200rpm overnight;

taking the culture solution of the single strain of the PigA gene exchange strain as a seed solution, inoculating the seed solution into an LC liquid culture medium according to the inoculation amount of 0.1%, and culturing for 24 hours in a constant temperature shaking table at 30 ℃ and 200 rpm;

diluting and coating the LC liquid culture medium cultured with the single-exchange strain of the pigA gene on an LCS plate, and culturing for 48 hours in a 30 ℃ thermostat;

selecting the recombinant bacterial colony on the LCS plate, carrying out PCR amplification on the recombinant bacterial colony by using detection primers UF and DR, detecting an amplification product by using agarose gel electrophoresis (shown in an attached figure 3), and finally screening to obtain a positive bacterial colony with the PigA gene knocked out, namely a PigA gene deletion strain;

wherein: the sequences of the detection primers UF and DR are as follows:

UF：5-TAACCATGTTACTGGTAACTGGAA-3

DR：5-CACCACAAAGACAGCCATCATCAA-3

the template DNA of PCR is DNA extracted from somatic cells;

the PCR amplification system is 20 μ L, and comprises:

2 XTaq DNA polymerase buffer 10. mu.L; upstream primer 0.3 mu mol/L; downstream primer 0.3 mu mol/L; 0.1 mu g of template DNA; the remaining volume was replenished with sterile double distilled water.

The reaction conditions for PCR amplification are as follows:

pre-denaturation at 94 ℃ for 10 min, after the pre-denaturation was complete the following procedure was carried out: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 1.5 minutes, and 30 cycles were repeated. After the circulation was completed, the temperature was 72 ℃ for 10 minutes.

The correct strain with the deletion of the pigA gene and the wild-type Serratia sp.ATCC 39006 are streaked on an LB plate in four regions, and cultured overnight in an incubator at 30 ℃ to observe colonies, so that the colonies of the strain with the deletion of the pigA gene are milk white and can not produce prodigiosin any more (figure 4), and the method disclosed by the embodiment can indeed knock out the pigA gene in the Serratia sp.ATCC 39006.

It will be apparent to those skilled in the art that the above description of specific embodiments of the invention is not intended to limit the application of the invention, and that various equivalents and modifications may be made thereto depending on the circumstances. All such substitutions and modifications are intended to be within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

<110> university of Jiangsu profession

<120> plasmid pMMB1 for enterobacter gene editing and construction method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

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<211> 5609

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tgccgcaagc actcagggcg caagggctgc taaaggaagc ggaacacgta gaaagccagt 60

ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa 120

aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac 180

tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag 240

gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc 300

aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 360

ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 420

caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 480

gttctttttg tcaagaccga cctgtccggt gccctgaatg aactccaaga cgaggcagcg 540

cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 600

gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 660

caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 720

cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 780

actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 840

gcgccagccg aactgttcgc caggctcaag gcgcggatgc ccgacggcga ggatctcgtc 900

gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 960

ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 1020

cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 1080

atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 1140

gcgggactct ggggttcgct agaggatcga tcctttttaa cccatcacat atacctgccg 1200

ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc 1260

aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag 1320

attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa 1380

agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta 1440

aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt 1500

gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact 1560

tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg 1620

agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta 1680

ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat 1740

ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac 1800

agcaaaaaaa tgaaaaatat caagtttctg aatttgattc gtccacaatt aaaaatatct 1860

cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg 1920

tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg 1980

atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga 2040

aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa 2100

aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt 2160

tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag 2220

ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa 2280

tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact 2340

acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca 2400

aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt 2460

tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac 2520

ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg 2580

agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag 2640

taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca 2700

ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc 2760

ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg 2820

tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac 2880

ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg 2940

cagacaaaca atcaacgttt gcgccgagct tcctgctgaa catcaaaggc aagaaaacat 3000

ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca 3060

aaagaaaatg ccgatgggta ccgagcgaaa tgaccgacca agcgacgccc aacctgccat 3120

cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc 3180

gggacgccct cgcggacgtg ctcatagtcc acgacgcccg tgattttgta gccctggccg 3240

acggccagca ggtaggccga caggctcatg ccggccgccg ccgccttttc ctcaatcgct 3300

cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct ggttggcttg 3360

gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg ccagcctcgc 3420

agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac 3480

ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa 3540

ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat 3600

accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa gcgctgcttc 3660

cctgctgttt tgtggaatat ctaccgactg gaaacaggca aatgcaggaa attactgaac 3720

tgaggggaca ggcgagagac gatgccaaag agctcctgaa aatctcgata actcaaaaaa 3780

tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc 3840

aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg 3900

atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc 3960

gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg 4020

cttctgtttc tatcagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat 4080

cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc 4140

caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg 4200

aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac 4260

ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag 4320

ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaaaagg 4380

atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4440

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga ctcgagtctg 4500

aagatcagca gttcaacctg ttgatagtac gtactaagct ctcatgtttc acgtactaag 4560

ctctcatgtt taacgtacta agctctcatg tttaacgaac taaaccctca tggctaacgt 4620

actaagctct catggctaac gtactaagct ctcatgtttc acgtactaag ctctcatgtt 4680

tgaacaataa aattaatata aatcagcaac ttaaatagcc tctaaggttt taagttttat 4740

aagaaaaaaa agaatatata aggcttttaa agcttttaag gtttaacggt tgtggacaac 4800

aagccaggga tgtaacgcac tgagaagccc ttagagcctc tcaaagcaat tttcagtgac 4860

acaggaacac ttaacggctg acatgggaat tctactgttt gggtgtatga gctatttgca 4920

aaggaaattg gtgatgacaa agctctcgag gagcctatgg aaaaacgcca gcaacgcggc 4980

ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 5040

ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 5100

ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa 5160

accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 5220

ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 5280

ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 5340

atttcacaca ggaaacagct atgacatgat tacgccaagc ttgcatgcct gcaggtcgac 5400

tctagaggat ccccgggtac cgagctcgaa ttcactggcc gtcgttttac aacgtcgtga 5460

ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 5520

ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 5580

tggcgaatgg cgataagcta gcttcacgc 5609

<210> 2

<211> 7658

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tgccgcaagc actcagggcg caagggctgc taaaggaagc ggaacacgta gaaagccagt 60

ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa 120

aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac 180

tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag 240

gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc 300

aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 360

ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 420

caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 480

gttctttttg tcaagaccga cctgtccggt gccctgaatg aactccaaga cgaggcagcg 540

cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 600

gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 660

caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 720

cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 780

actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 840

gcgccagccg aactgttcgc caggctcaag gcgcggatgc ccgacggcga ggatctcgtc 900

gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 960

ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 1020

cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 1080

atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 1140

gcgggactct ggggttcgct agaggatcga tcctttttaa cccatcacat atacctgccg 1200

ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc 1260

aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag 1320

attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa 1380

agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta 1440

aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt 1500

gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact 1560

tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg 1620

agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta 1680

ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat 1740

ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac 1800

agcaaaaaaa tgaaaaatat caagtttctg aatttgattc gtccacaatt aaaaatatct 1860

cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg 1920

tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg 1980

atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga 2040

aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa 2100

aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt 2160

tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag 2220

ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa 2280

tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact 2340

acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca 2400

aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt 2460

tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac 2520

ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg 2580

agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag 2640

taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca 2700

ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc 2760

ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg 2820

tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac 2880

ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg 2940

cagacaaaca atcaacgttt gcgccgagct tcctgctgaa catcaaaggc aagaaaacat 3000

ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca 3060

aaagaaaatg ccgatgggta ccgagcgaaa tgaccgacca agcgacgccc aacctgccat 3120

cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc 3180

gggacgccct cgcggacgtg ctcatagtcc acgacgcccg tgattttgta gccctggccg 3240

acggccagca ggtaggccga caggctcatg ccggccgccg ccgccttttc ctcaatcgct 3300

cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct ggttggcttg 3360

gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg ccagcctcgc 3420

agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac 3480

ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa 3540

ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat 3600

accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa gcgctgcttc 3660

cctgctgttt tgtggaatat ctaccgactg gaaacaggca aatgcaggaa attactgaac 3720

tgaggggaca ggcgagagac gatgccaaag agctcctgaa aatctcgata actcaaaaaa 3780

tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc 3840

aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg 3900

atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc 3960

gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg 4020

cttctgtttc tatcagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat 4080

cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc 4140

caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg 4200

aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac 4260

ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag 4320

ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaaaagg 4380

atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4440

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga ctcgagtctg 4500

aagatcagca gttcaacctg ttgatagtac gtactaagct ctcatgtttc acgtactaag 4560

ctctcatgtt taacgtacta agctctcatg tttaacgaac taaaccctca tggctaacgt 4620

actaagctct catggctaac gtactaagct ctcatgtttc acgtactaag ctctcatgtt 4680

tgaacaataa aattaatata aatcagcaac ttaaatagcc tctaaggttt taagttttat 4740

aagaaaaaaa agaatatata aggcttttaa agcttttaag gtttaacggt tgtggacaac 4800

aagccaggga tgtaacgcac tgagaagccc ttagagcctc tcaaagcaat tttcagtgac 4860

acaggaacac ttaacggctg acatgggaat tctactgttt gggtgtatga gctatttgca 4920

aaggaaattg gtgatgacaa agctctcgag gagcctatgg aaaaacgcca gcaacgcggc 4980

ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 5040

ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 5100

ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa 5160

accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 5220

ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 5280

ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 5340

atttcacaca ggaaacagct atgacatgat tacgccaagc ttgcatgcct gcagctatcg 5400

gttactcacg caacatggct tccgcgcccg gaatttgtcc gggggatata aaacttacag 5460

cctgacgttg tccgcctgaa ccattagcgt tcggattaat cattggcatg atggcaagta 5520

taggggccgt catgccaaac cagatgatgt aatataaaaa tacgatttca ctatgtgagt 5580

gcaataaccg gtatcctttc gcttgccgcc atcttgcaaa taaagttatc caggacatct 5640

atattcatta ttatgtaata tgcttattta ttcaatcttt cgagcattga gtaattactc 5700

ttgtgtcaat attgtatttt attattagat gaggttattt attttattgt tctaacttcg 5760

gaatttattg aactataagt ttaatgatat tgtaatattt aaataatata tggcagtaag 5820

gttttgtata aaacacaata ttaattcaac tgatcagtta accctttatt caggaaaaaa 5880

ctcacgcata ggaacaccat aatctataaa tttatatcat gatatggtca cggtttgcct 5940

cgatacacca attcttcttc accaaattgt aacaataagc ttaatgaatc cctagctgat 6000

gaccttcggt catcagtgtc gttttagcgt cggtttaaca atggcctcta tttacttgca 6060

ataaaaatac ttagctaaat caatattaaa agtgcatcaa tattctcact gctccgtaag 6120

tcagaacaac gtcaaacgct tatgcaagat ctggattgtt taatgtttgt ttattaaaag 6180

tgtaatttgt aaaatattgt gtattgttgt tttattaaaa atttattata tatcaaaggt 6240

taaaccatgt gttaattgtg ggtatgtaac catgttactg gtaactggaa agctattcac 6300

attacgacgt attttccaat gttgcatttt gtgctcatca acattaaaga taatagcgaa 6360

gtcaattaac cgttgtagca atggaagcaa tggagtgttt tatggatttt aacctgtcaa 6420

atagtcagga attcggaaca tcatcatgat tattcaacgg ctcttcggca tcctctatat 6480

gcttgccggt ttggcgaaag ctttcccgca gtttgagaat gttcccgctg ttcttcggca 6540

ggcggcaatt gccaaccagg gaacctggta cgcggcggcg agtatctggc tgggcgcaca 6600

tggtgatgtg atcaatatcc tggtgggagt ggtgctgttt gggtcgggag tgatattaat 6660

gctcaaccca ctctggacga cgctggtgat ctacgcccag cttttgatga tggctgtctt 6720

tgtggtgatt ttgcatcagt cccagcccca ggtcatgttg ctggatggcg tatttgcgct 6780

ggccgcgctt tacatgctgc gcggtcagta tcaccgtaag cctaagccga gaaccttccc 6840

gaccacgtct ttttcgctgc ccacaccctc ttctgaatca tcgttttctg ctcccttggg 6900

tgatgagtat gatgtcgtga ttatcggcgg tggtgcctcc ggactgacgg cggccagtga 6960

atttactcat gaacgggtgt tggtgcttga aaaaagctcc actttcgggg ggaatgcccg 7020

ctaccacacc tttaaccggt taaagcatcc caccgccggt gtttgttttc aagaaccgtt 7080

tcccggttcg aatatgctgc gcttactgaa aaagattggt ctggagggaa aatacaagtc 7140

caacgaaaag gacacgctgg tcttttttga taccttttta ttgctcaaat gcttaggtga 7200

aattgtggtc ggctttatta agcaaccacg ttatctgctc aaactctcgg tctgggggct 7260

gaccagccag ctttttctgc atgcgataat tggtaaaccc tacgtggtag cggccaaaca 7320

acttggtgac ccgatctttg ccgatcttta tacctttctg gataagttct ctcctcgtgg 7380

cgacttttat ccacgcctgc cctggacacc gaacggttcc tggagcaaag cgcatatgga 7440

gttgctcgat aatatggatc cccgggtacc gagctcgaat tcactggccg tcgttttaca 7500

acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 7560

tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 7620

cagcctgaat ggcgaatggc gataagctag cttcacgc 7658

Claims (6)

1. A plasmid pMMB1 has a nucleotide sequence shown in SEQ ID NO. 1.

2. A plasmid, pMMB1, characterized by: the nucleotide sequence of the gene is a kanamycin resistance gene and a promoter region thereof from the 208-1140-site deoxyribonucleotide at the 5' end; the 1192-position 3053 deoxyribonucleotide is a fructan sucrase gene sacB and a promoter region thereof; 3407-3516 shows that the deoxyribonucleotide is an IncP group plasmid conjugation transfer site; the 4497-4944 deoxyribonucleotide is a lambda pir protein-dependent replicon oriR 6K; 5287-5586-position deoxyribonucleotide is lacZ alpha gene for screening blue white spots and a promoter region thereof; the 5383-5417-site deoxyribonucleotide is a multiple cloning site for inserting an exogenous fragment.

3. The method of construction of plasmid pMMB1 of claim 1 or 2, characterized in that: the method comprises the following steps:

(1) preparation of the cleavage product of the lambda pir protein-dependent replicon oriR 6K:

(1.1) carrying out PCR amplification on the lambda pir protein-dependent replicon oriR6K by using the plasmid pKNG101 as a template to obtain an amplification product; recovering the amplification product using a universal nucleic acid recovery kit to obtain a lambda pir protein-dependent replicon oriR 6K;

(1.2) treating the recovered amplification product with restriction endonuclease XhoI to obtain a lambda pir protein-dependent replicon oriR6K enzyme-cleaved product, and recovering the product by using a general-purpose nucleic acid recovery kit;

(2) preparation of plasmid backbone digestion product:

(2.1) PCR-amplifying sequences except the replicon by using an upstream primer DoriF and a downstream primer DoriR with the plasmid pK19mobsacB as a template to obtain an amplification product. Recovering the amplification product by using a universal nucleic acid recovery kit to obtain a plasmid skeleton;

(2.2) treating the recovered amplification product with restriction endonuclease XhoI to obtain a plasmid backbone enzyme digestion product, and recovering the enzyme digestion product by using a universal nucleic acid recovery kit;

(3) preparation of plasmid pMMB 1:

and connecting the lambda pir protein dependent replicon oriR6K enzyme digestion product with the plasmid skeleton enzyme digestion product to obtain the pMMB1 plasmid.

4. The method of construction of plasmid pMMB1 of claim 3, wherein: in the step (1.1), the amplification primers in the PCR amplification system are as follows:

the upstream primer R6 KF: 5-CCGCTCGAGTCTGAAGATCAGCAGTTCAACCTG-3;

the downstream primer R6 KR: 5-CCGCTCGAGAGCTTTGTCATCACCAATTTCCTT-3;

the PCR amplification system is 25 μ L, and comprises:

10 XDNA polymerase buffer 2.5. mu.L; 0.2mmol/L dATP; 0.2mmol/L dTTP; 0.2mmol/L dCTP; 0.2mmol/L dGTP; the upstream primer R6KF 0.3 is 0.3 mu mol/L; the downstream primer R6KR 0.3 is 0.3 mu mol/L; 0.1 mu g of template DNA; KOD DNA polymerase 0.5U, magnesium ion 1.5mmol/L, make up the remaining volume with sterilized double distilled water;

the reaction conditions for PCR amplification are as follows:

pre-denaturation at 98 ℃ for 5 min, after the pre-denaturation had ended the following procedure was carried out: denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 30 seconds, extension at 68 ℃ for 20 seconds, repeating for 30 cycles; after the circulation was completed, the temperature was 68 ℃ for 10 minutes.

5. The method of construction of plasmid pMMB1 of claim 3, wherein: in the step (2.1), the amplification primers in the PCR amplification system are as follows:

DoriF：5-CCGCTCGAGGAGCCTATGGAAAAACGCCAGCAACGCGGC-3；

DoriR：5-CCGCTCGAGTCTCAAGAAGATCCTTTGATCTTTTCTACG-3；

the PCR amplification system is 25 μ L, and comprises:

10 XDNA polymerase buffer 2.5. mu.L; 0.2mmol/L dATP; 0.2mmol/L dTTP; 0.2mmol/L dCTP; 0.2mmol/L dGTP; upstream primer DoriF 0.3 mu mol/L; downstream primer DoriR 0.3 mu mol/L; 0.1 mu g of template DNA; KOD DNA polymerase 0.5U, magnesium ion 1.5mmol/L, make up the remaining volume with sterilized double distilled water;

the reaction conditions for PCR amplification are as follows:

pre-denaturation at 98 ℃ for 5 min, after the pre-denaturation had ended the following procedure was carried out: denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 30 seconds, extension at 68 ℃ for 3 minutes, and 30 cycles were repeated. After the circulation was completed, the temperature was 68 ℃ for 10 minutes.

6. Use of the plasmid pMMB1 of claim 1 or 2 for gene editing.

Images