CN101250548B - Shuttle plasmid and derivative plasmid thereof - Google Patents

Abstract

The invention discloses a shuttle plasmid and a derivative plasmid thereof. A nucleotide sequence of the shuttle plasmid which is provided by the invention is a sequence 1 in a sequence table and a 4872-5664th deoxyribonucleotide from 5'end of the sequence 1 in the sequence table is a kanamycin resistance gene and a promoter thereof. The invention also discloses the derivative plasmid thereof, which is obtained through inverting the kanamycin resistance gene of the 4872-5664th deoxyribonucleotide from the 5'end of the sequence 1 in the sequence table and the promoter of the kanamycin resistance gene and through inserting transcription terminators of a reporter gene and a corynebacterium glutamate LeuB gene in multiple cloning sites, the direction of the reporter gene is opposite to the direction of the kanamycin resistance gene and the promoter of the kanamycin resistance gene, and the transcription terminators of the corynebacterium glutamate LeuB gene are located between the reporter gene and the kanamycin resistance gene. The two plasmids have high stability and can obtain wide application on stic engineering.

Description

A kind of shuttle plasmid and the plasmid of deriving thereof

Technical field

The present invention relates to a kind of shuttle plasmid and the plasmid of deriving thereof.

Background technology

Plasmid is an extra-chromosomal genetic element, can carry out self-replicating.It is not the essential genetic elements of host cell vital movement, but can give host cell some special nature, and resistance is for example degraded or utilized some compound etc.In molecular biology research, plasmid can be used as carrier and is used to clone foreign gene.Research about plasmid is quite deep, and people constantly find novel plasmid from nature on the one hand, also constantly makes up the plasmid of deriving on the other hand on the basis of existing plasmid, as engineered carrier.

In bacterial gene engineering field, most plasmid vectors are from the Gram-negative bacteria intestinal bacteria, many commercializations.To the research of gram-positive microorganism Corynebacterium glutamicum plasmid begin later.1984, Corynebacterium glutamicum plasmid pBL1, pCG1, pCG2 and pCG4 etc. have successively been reported.(Santamaria.R.et al 1984J.Gen.Microbiol.130.2237-2246.Ozaki.A et al 1984 Mol.Gen.Genet 196.175-178 Katsumata.R.et al 1984 J.Bacteriol.159.306-311) have isolated 24 kinds of endogenous plasmids at present from various excellent bacillus.Most of miniplasmids is a hidden-type in these 24 kinds of endogenous plasmids, and only plasmid pXZ10145 is an exception, and this plasmid has a chloramphenicol resistance gene.(1993 biotechnology journal 9.216-222 such as Shen Tianxiang) and in bigger plasmid also have only pAG1, pCG4 and pTET3 coding antibiotics resistance gene.According to possible copy mode of Corynebacterium glutamicum and the similarity between the replication initiator protein, can be divided into two groups to them.One group is to take to roll the ring mode to duplicate, and comprises pBL1 family plasmid and pCG1 family plasmid; Another group takes the theta replication mode to duplicate, and comprises pXZ10145 family plasmid and pCRY4 family plasmid.In general, the plasmid of taking the theta replication mode to duplicate takes the plasmid of rolling-circle replication to have advantages of higher stability.In the Corynebacterium glutamicum gene engineering research, the cloning vector major part that has made up comes from hidden-type plasmid pBL1 (Miwa.K et al1985 Gene 39.281-286; Yeh.P et al 1986 Gene 47 301-306; Santamari.R.I.1987Gene 56 199-208; Patek.M.1989 Appl.Microbiol.Biotechnol.31.65-69; Nesvera.J.et al 1990 Folia Microbiol.35.273-277; Mukherjee.K.J.et al1990.J.Biotechnol.16.109-122; Eikmanns.B.J.et al 1991 Gene 102.93-98; Tauch.A.et al 1998a Arch.Microbiol.169.303-312; ); PCG1 (Miwa.K.et al 1985 Gene 39.281-286; Yoshihama.M.et al 1985.J.Bacteriol.162.591-597; ); PCG2 (Ozaki.A et al 1984.Mol.Gen.Genet 196.175-178; Ikeda.M.and Katsumata.R.1999.Appl.Environ.Microbial.65.2497-2502; ) and .pCG4 (Ikeda.M.and Katsumata.R.1999.Appl.Environ.Microbial.65.2497-2502; ).The promotor of the resistant gene of kantlex, bleomycin, paraxin and erythromycin all can be widely used in vector construction by the archaeal dna polymerase of excellent bacillus identification.On the other hand, because colibacillary genetic manipulation system is comparative maturity, the most cloning vectors that use in the Corynebacterium glutamicum gene engineering research at present all are all reproducible shuttle vectorss between intestinal bacteria and Corynebacterium glutamicum, and have multiple clone site.But rarely has the report that makes up cloning vector from Corynebacterium glutamicum plasmid at present with theta replication mechanism.

Summary of the invention

The purpose of this invention is to provide a kind of shuttle plasmid and the plasmid of deriving thereof.

A kind of shuttle plasmid provided by the present invention, name is called pAK6, and its nucleotide sequence is the sequence 1 in the sequence table.

PAK6 is the shuttle vector that can duplicate in Corynebacterium glutamicum and intestinal bacteria.

Wherein, pAK6 comprises following tactic multiple clone site a: KpnI-SacI-EcoRI-BglII-HindIII-PstI-XbaI-SphI; The nucleotide sequence of described multiple clone site be in the sequence table sequence 1 from shown in the deoxyribonucleotide of 5 ' terminal 14-48 position.Except that described multiple clone site, pAK6 also comprises an ampicillin resistance gene and promotor thereof and kalamycin resistance gene and promotor thereof; The nucleotide sequence of described ampicillin resistance gene and promotor thereof be in the sequence table sequence 1 from shown in the deoxyribonucleotide of 5 ' terminal 2085-1227 position; The nucleotide sequence of described kalamycin resistance gene and promotor thereof be in the sequence table sequence 1 from shown in the deoxyribonucleotide of 5 ' terminal 4872-5664 position.

The present invention also provides by pAK6 deutero-shuttle plasmid pAKC6, and this shuttle plasmid can be used as the promoter detection carrier.

PAKC6 provided by the present invention, be kalamycin resistance gene and promoter fragment thereof with pAK6, be that sequence 1 inserts in its multiple clone site that reporter gene and Corynebacterium glutamicum LeuB gene transcription terminator obtain in the sequence table after 5 ' terminal 4872-5664 position deoxyribonucleotide sequence is carried out inversion; The direction of described reporter gene is opposite with described kalamycin resistance gene and promotor direction thereof; Described Corynebacterium glutamicum LeuB gene transcription terminator is between described reporter gene and described kalamycin resistance gene.

Wherein, described reporter gene can be a chloramphenicol acetyl transferasegene; The nucleotide sequence of described chloramphenicol acetyl transferasegene be in the sequence table sequence 2 from shown in the deoxyribonucleotide of 5 ' terminal 50-827 position.The nucleotide sequence of described LeuB gene transcription terminator be in the sequence table sequence 2 from shown in the deoxyribonucleotide of 5 ' terminal 6-37 position.

The nucleotide sequence of plasmid pAKC6 specifically can be the sequence 2 in the sequence table.

Plasmid pAK6 of the present invention and pAKC6 can use in genetically engineered.

The plasmid stability experimental result shows, plasmid pAK6 and pAKC6 that the present invention makes up are not having under the situation of selective pressure, 100 generations of cultured continuously, plasmid pAK6 conservation rate is 100%, plasmid pAKC6 conservation rate is more than 98.5%, compares apparently higher than rolling-circle replication type plasmid with the conservation rate (below 28%) of rolling-circle replication type plasmid pUL340; The Electronic Speculum test experience of plasmid replication intermediate shows that plasmid pAK6 and pAKC6 duplicate with the θ type; The Electronic Speculum test experience of plasmid stability experiment and plasmid replication intermediate is the stability height of consolidated statement phaneroplasm grain pAK6 and pAKC6 as a result, will be widely used on genetically engineered.

Description of drawings

Fig. 1 is the physical map of plasmid pAK6.

Fig. 2 is the physical map of plasmid pAKC6.

Fig. 3 is the design of graphics of plasmid pAK6.

Fig. 4 is the restriction enzyme digestion and electrophoresis analysis of plasmid pAK6

M：DNA?marker、λ/EcoRT?14I、1：EcoR?I、2：Kpn?I、3：Bgl?II、4：Hind?III、5：Pst?I、6：Xba?I、7：Sph?I。

Fig. 5 is a pcr amplification chloramphenicol acetyl transferasegene gene

M:DNA marker is λ/EcoT14I, a 1:PCR amplification chloramphenicol acetyl transferasegene product.

Fig. 6 is the pcr amplification kalamycin resistance gene

M:DNA marker is λ/EcoT14I, a 1:PCR amplification kalamycin resistance gene product.

Fig. 7 is the design of graphics of plasmid pAKC6.

Fig. 8 is the restriction enzyme digestion and electrophoresis analytical results of plasmid pAKB6 and pAKC6.

M:DNA marker is λ/EcoT14I, 1:pAKB6/NcoI, 2:pAKC6/NcoI

Fig. 9 is the restriction enzyme digestion and electrophoresis analytical results of plasmid pAKC6.

M：DNA?marker、λ/EcoRT?14I、1：BamHI-KpnI、2：HindIII-XbaI。

Figure 10 is pUL340/13032 stability experiment result

Figure 11 is the pAK6/13032 stability experiment

Figure 12 is the pAKC6/13032 stability experiment

Figure 13 is a plasmid pAK6 replicative intermediate electromicroscopic photograph

Figure 14 is a plasmid pAKC6 replicative intermediate electromicroscopic photograph

Embodiment

The structure of embodiment 1, plasmid pAK6

The building process of plasmid pAK6 as shown in Figure 3, concrete grammar is as follows:

Be the plasmid that sets out with Corynebacterium glutamicum plasmid pXZ10145 (1993 biotechnology journal 9.216-222 such as Shen Tianxiang) (Institute of Microorganism, Academia Sinica), with EcoR I/SpeI double digestion plasmid pXZ10145, separate through agarose gel electrophoresis, reclaim the big fragment that has replication origin.With EcoR I/Xba I double digestion escherichia coli plasmid pUC119-KanR (Guoqing Niu et al 2006 Metabolic Engineering8.183-195) (Institute of Microorganism, Academia Sinica), reclaim the small segment that has the KanR gene.Two fragments are connected construction recombination plasmid.With recombinant plasmid transformed Corynebacterium glutamicum ATCC 13032, extract plasmid, called after pAK0.For on pAK0, introducing the intestinal bacteria replication origin,, reclaim the big fragment that has excellent bacillus replication origin and KanR gene with EcoR I/AatII double digestion pAK0; Escherichia coli plasmid pUC19 is carried out same double digestion, reclaim the big fragment that has the escherichia coli plasmid replication origin, two big fragments are connected construction recombination plasmid.With the recombinant plasmid transformed bacillus coli DH 5 alpha, extract recombinant plasmid, obtain having the plasmid of Corynebacterium glutamicum plasmid replication starting point and escherichia coli plasmid replication origin and kalamycin resistance gene and ampicillin resistance gene, called after pAK2.Because going up the multiple clone site district, pAK2 also has other EcoR I, Kpn I, BamH I restriction enzyme site, be unfavorable for cloning the external source fragment, so redesigned the multiple clone site district, particularly introduced Bgl II and singly cut the site, convenient segmental clone through Sau3A I complete digestion.Obtain the multiple clone site district by the following method: design two oligonucleotide sequences, be respectively S1 and S2, the sequence of S1 and S2 is as follows: 5 ' AGCTGCATGCTCTA GACTGCAGAAGCTT AGAT CTG-3 ' and 5 '-AATTCAGATCTAAGCTTCTGCAGTC TAGAGC AT G C-3 '.It is 50mmol/L that synthetic S1 and S2 oligonucleotide sequence are made its concentration with the sterile distilled water dissolving respectively, with the two mixing, kept 5-60 minute at 60 ℃-90 ℃, lower the temperature, obtain having the nucleotide fragments of multiple clone site district (MCS), this fragment has Bgl II, HindIII, Pst I, Xba I, the single restriction enzyme site of Sph I, two ends be respectively EcoR I sticking end and with Hind III complementary overhang.With HindIII/EcoR I double digestion pAK2, reclaim big fragment, be connected with the nucleotide fragments that has the multiple clone site district, construction recombination plasmid, this recombinant plasmid transformed bacillus coli DH 5 alpha extracts recombinant plasmid, called after pAK6.This plasmid has the multiple clone site district of arranging in the following order: KpnI-SacI-EcoRI-BglII-HindIII-PstI-XbaI-SphI.The pAK6 electric shock is transformed Corynebacterium glutamicum ATCC 13032, extract the pAK6 plasmid, again transformed into escherichia coli DH5 α, extract the pAK6 plasmid, carry out the restriction enzyme digestion and electrophoresis checking, the result conforms to the plasmid that changes over to, illustrates that plasmid pAK6 can shuttle back and forth between intestinal bacteria-Corynebacterium glutamicum.Use EcoR I, Kpn I, Bgl II, Hind III, Pst I, Xba I and Sph I digested plasmid pAK6 respectively, each enzyme has only a restriction enzyme site thereon respectively, and the result conforms to expection as shown in Figure 4.Sequencing result shows that the size of plasmid pAK6 is 5684bp, and its nucleotide sequence is shown in the sequence in the sequence table 1.Plasmid pAK6 belongs to less a kind of in the shuttle vectors of having reported at present.Utilize its cloning site that has, pAK6 can be transformed into integrative vector easily, expression vector, promotor carrier detection etc.

The structure of embodiment 2, plasmid pAKC6

The building process of plasmid pAKC6 as shown in Figure 7, concrete grammar is as follows:

For making up the promotor carrier detection, on plasmid pAK6, introduce reporter gene chloramphenicol acetyl transferasegene CAT and Corynebacterium glutamicum LeuB gene transcription terminator T _LcuBAt first by coding region and the SD sequence of PCR method from plasmid pXC99E (Kirchner O and Tauch A 2003 J.Biotechnology 104:287-299) (Institute of Microorganism, Academia Sinica) amplification chloramphenicol acetyl transferasegene (GenBank No.AT219685).The primer is

P1:5 '-GGG AAGCTTAAGTAATTAACAGGAGCTAAGGAAGCTAAA-3 ' and

P2:5 '-AAC TCTAGATAAGGGATTTTGGTCATGCG-3 ', underscore partly are respectively the recognition site of Hind III and Xba I.

Reaction system is: cumulative volume 50 μ L, 2 * GC bufferII, 25 μ L, dNTP 7 μ L, LA Taq archaeal dna polymerase 0.5 μ L, each 1 μ L of 50 μ mol/L primers, dna profiling 1 μ L, ddH ₂O 15 μ L.

PCR reaction conditions: 94 ℃ of 2min at first; 94 ℃ of 30s, 55 ℃ of 45s, 72 ℃ of 1min, 30cycles then; 72 ℃ of 5min again.The electrophoresis result of pcr amplification CAT gene product as shown in Figure 5.

Then, according to document (M.Pate k.et al 1998 Appl.Microbiol.Biotechnol.50.42-47) design oligonucleotides sequence S3 and S4, S3 and S4 sequence are as follows:

5-AATTCAAAGTAA ACCCCTCGCCATAAAA GGCGAGGGGTAC-3 ' and 5 '-CCCTCGCCTTTTATGGCGA GG GGTTTACTTTG-3 '; Synthetic S3 and S4 oligonucleotide fragment are mixed with 50mmol/L solution, with the two mixing, kept 5-60 minute cooling at 60 ℃-90 ℃, must arrive the fragment that two ends are respectively the sticking end of EcoR I and Kpn I, i.e. Corynebacterium glutamicum LeuB gene terminator sequence.

Plasmid pAK6 is carried out double digestion with EcoR I/Kpn I, reclaim big fragment, with Corynebacterium glutamicum LeuB gene terminator T _LeuBConnect construction recombination plasmid transformed into escherichia coli DH5 α, recombinant plasmid called after pAKT6.Further pAKT6 is carried out double digestion with HindIII/Xba I, reclaim big fragment; With the two chloramphenicol acetyl transferasegenes (CAT) of cutting of same enzyme, two fragments are connected, construction recombination plasmid transformed into escherichia coli DH5 α extracts recombinant plasmid, called after pAKB6.Because the KanR gene promoter on the pAKB6 is stronger, and itself does not have the terminator sequence, thereby might read over terminator T _LeuBFor avoiding this phenomenon, adopt round pcr that the KpnI of KanR gene both sides and BamHI site are exchanged.Method is as follows: according to a pair of primer P3 of sequences Design and the P4 of plasmid pUC119-KanR (Guoqing Niu et al 2006Metabolic Engineering 8.183-195) (Institute of Microorganism, Academia Sinica), the sequence of primer P3 and P4 is as follows: 5 '-TAGA GGTACCCCTGGATACCGCTCG-3 ' and 5 '-GCTC GGATCCCGAACCCCAGAGTCC-3 ', underscore partly are Kpn I and BamH I recognition site, and making the original BamH I in KanR gene both sides, Kpn I site rite-directed mutagenesis is Kpn I, BamH I, to change the direction of insertion of KanR gene in pAKB6.With plasmid pUC119-KanR (Guoqing Niu et al 2006 MetabolicEngineering 8.183-195) (Institute of Microorganism, Academia Sinica) is template, carries out pcr amplification with primer P3/P4.Reaction system is: cumulative volume 50 μ L, 2 * GC bufferII, 25 μ L, dNTP 7 μ L, LA Taq archaeal dna polymerase 0.5 μ L, each 1 μ L of 50 μ mol/L primers, dna profiling 1 μ L, ddH ₂O 15 μ L.

PCR reaction conditions: 94 ℃ of 2min of elder generation; 94 ℃ of 30s, 55 ℃ of 45s, 72 ℃ of 1min, 30 cycles again; Last 72 ℃ of 5min.The electrophoresis result of pcr amplification KanR gene product as shown in Figure 6.

The product of Kpn I/BamH I double digestion pcr amplification KanR gene reclaims the KanR gene fragment.The two enzyme plasmid pAKB6 of KpnI/BamHI reclaim the big fragment of pAKB6.The KanR gene fragment is connected with the big fragment of pAKB6, construction recombination plasmid, transformed into escherichia coli DH5 α extracts recombinant plasmid, carries out enzyme with NcoI, BamHI/KpnI and HindIII/XbaI and cuts evaluation, and the result conforms to expection.Result such as Fig. 8 and shown in Figure 9.This plasmid called after pAKC6.KanR genetic transcription direction on the pAKC6 and CAT transcriptional orientation opposite can not cause and read over.The pAKC6 electric shock is transformed Corynebacterium glutamicum ATCC 13032, extract the pAKC6 plasmid, transformed into escherichia coli DH5 α extracts the pAKC6 plasmid again, analyzes through NcoI, BamHI/KpnI and HindIII/XbaI restriction enzyme digestion and electrophoresis, and the result conforms to the plasmid that changes over to.Illustrate that pAKC6 can shuttle back and forth between Corynebacterium glutamicum and intestinal bacteria.

Sequencing result shows that the size of plasmid pAKC6 is 6474bp, has sequence shown in the sequence 2 in the table.Plasmid pAKC6 is a reporter gene with CAT, can be used as the intensity that shuttle vectors is used for screening promotor and measures promotor.

Embodiment 3, plasmid pAK6 and pAKC6 adopt the θ mode to duplicate

1. the stability experiment of plasmid pAK6 and pAKC6

Plasmid pUL340 is a rolling-circle replication type plasmid, is contrast with plasmid pUL340, measures the stability of plasmid pAK6 and pAKC6.Method is as follows:

1) with the host of Corynebacterium glutamicum ATCC 13032 as plasmid pUL340 (Ramon I.Santamaria et al1985 J.Bacteriology Vol.162 No.1.p.463-467) (Institute of Microorganism, Academia Sinica), pAK6 and pAKC6.

PUL340, pAK6 and pAKC6 are imported respectively among the Corynebacterium glutamicum ATCC 13032, obtain containing pUL340 reorganization bacterium pUL340/13032, contain the reorganization bacterium pAK6/13032 of pAK6 and contain the reorganization bacterium pAKC6/13032 of pAKC6.

Single bacterium colony of pUL340/13032, pAK6/13032 and pAKC6/13032 is inoculated in the LB substratum that contains 50 μ g/ml kantlex 30 ℃ respectively is cultured to logarithmic phase, with mass percent be 0.85% physiological saline according to 10 times of gradient dilutions behind certain multiple, get 50 μ l and be coated on the LB flat board that does not contain kantlex, 30 ℃ cultivate the tally sheet colony number in order to calculate for the time.Dai Shike is calculated by following formula:

$G=\frac{t_1-{\mathrm{<figure-callout\; id="0"\; label="t"\; filenames="H2008100844866E00053.png,H2008100844866E00054.png"\; state="\{\{state\}\}">t</figure-callout>}}_0}{3.3\&times;\lg \frac{y}{x}}$

G be for the time, x is time t ₀The time the bacterium number, y is time t ₁The time the bacterium number.

The result shows under 30 ℃ of culture condition, Corynebacterium glutamicum pUL340/13032 for the time be 62 minutes; Corynebacterium glutamicum pAK6/13032 for the time be 83 minutes; Corynebacterium glutamicum pAKC6/13032 for the time be 83 minutes.

2) with 1: 60 volume ratio with 10 ⁵-10 ⁶Diluent be transferred to the 3ml LB that does not contain kantlex and shake 30 ℃ of cultivations in the pipe, dilution after 24 hours, the coated plate of repeating step 1 and switching are until cultivating about 100 generations.

Wherein, algebraically is measured as follows:

$n=\frac{\mathrm{lgy}-\mathrm{<figure-callout\; id="2"\; label="lgx\; lg"\; filenames="H2008100844866E00051.png,H2008100844866E00052.png"\; state="\{\{state\}\}">lgx</figure-callout>}}{\lg }$

Wherein n is an algebraically, and x is time t ₀The time the bacterium number, y is time t ₁The time the bacterium number.

3) get on the LB flat board that does not contain Kan in 200 steps 2 single bacterium colony respectively dibbling containing 50 μ g/mlKan and do not containing on the LB flat board of Kan, deposit viable count on the LB flat board of depositing viable count/do not contain Kan on the LB flat board of the conservation rate of plasmid under the antibiotic-free selection condition=contain 50 μ g/mlKan.Do not having under the condition of selective pressure, in 100 generations of cultured continuously, the conservation rate of plasmid pUL340 is below 28%; The conservation rate of plasmid pAK6 is 100%; The conservation rate of plasmid pAKC6 is more than 98.5%.

Show that plasmid pAK6, pAKC6 stability are better than plasmid pUL340, plasmid pAK6, pAKC6 meet theta replication type plasmid than the stable rule of rolling-circle replication type plasmid (plasmid pUL340).

2. the Electronic Speculum of plasmid replication intermediate detects

In order to observe the copy mode of pAK6, pAKC6, pAK6, pAKC6 are imported the reorganization bacterium pAKC6/ATCC 13032 that obtains containing the reorganization bacterium pAK6/ATCC 13032 of pAK6 and contain pAKC6 among the Corynebacterium glutamicum ATCC13032.Reorganization bacterium pAK6/ATCC 13032 and pAKC6/ATCC 13032 are inoculated in respectively in the LB substratum that contains 50 μ g/ml kantlex, are cultured to logarithmic phase.Extract plasmid pAK6 and pAKC6:Mojica F.J.M.et al 1994 J.Bacteriologyvol.176.No.16.p.4966-4973 according to the gentle method in the following document.To extract plasmid pAK6 and pAKC6 and be dissolved in respectively in TE (the 10mMTris-HCl pH8.0 1mM EDTA) solution, final concentration is 10 μ g/ml.Use electron microscopic observation plasmid pAK6, pAKC6:Burkardt H, Lurz R. (1984) Electron microscopy.In P ü hler A﹠amp according to following document; Timmis KN. (eds), Advancedmolecular genetics.Springer-VerlagPress, Berlin, Heidelberg, New York and Tokyo, Chap.6, pp.286-288.

Concrete grammar is as follows:

Agents useful for same comprises, last phase: 2 μ l carbonate buffer solutions (400 μ l ddH ₂O, 40 μ l Na ₂CO ₃(1M) 20 μ l EDTA (02M)), 5 μ l methane amides, 1 μ l cytochrome C (1mg/ml), 2 μ l plasmid DNA (10 μ g/ml), pH10.5.On mutually now with the current, the time spent is placed on ice.Following phase: distilled water.

Step is as follows:

1, gets the culture dish of a cleaning, pour the distilled water (protruding an obvious convex surface is advisable) of capacity into, scrape the other end from culture dish one end gently with clean PVC then, as following phase;

2, put into a clean glass slide gently, an end leans against on the PVC, and an end is immersed in down below the liquid level of phase;

3, at following phase surface spray one deck talcum powder;

4, will drop in mutually on the slide glass of step 2 on the 5-20 μ l, make mutually and slowly flow down the formation unimolecular layer, left standstill 3-5 minute;

5, use the copper mesh that is loaded with pyroxylin (e) cement to dip in and get unimolecular layer, copper mesh is soaked 20 seconds (2 times), seasoning on filter paper then in 100% alcohol;

6, copper mesh is put into vacuum plating unit, and with the platinum iridium alloy spraying, projected angle is 70, and the universal stage rotating speed is 90 rev/mins; Transmission electron microscope Hitachi H-700A observes.

The result shows that Figure 13 and Figure 14 are respectively the electromicroscopic photograph of plasmid pAK6 and pAKC6 replicative intermediate shown in Figure 13,14, can see having replication bubble in the replicative intermediate, illustrates that plasmid pAK6 and pAKC6 copy mode are the θ type.

Embodiment 4, with pAKC6 screening Corynebacterium glutamicum promotor

Will be through Corynebacterium glutamicum 10147 (the 2007 microorganism journals 47 (2) such as Li Kai: 191-196) (Institute of Microorganism, Academia Sinica) genomic DNA fragment of Sau3A I complete digestion, be connected the acquisition recombinant plasmid with the pAKC6 that cuts with Bgl II enzyme, with the recombinant plasmid transformed bacillus coli DH 5 alpha, obtain 20 transformants; In the LB flat board that contains 400 μ g/mL paraxin, there are 5 can grow the transformant dibbling; Extract recombinant plasmid, electric shock transforms Corynebacterium glutamicum 10147 competent cells respectively, cell after the conversion is coated on the LB flat board that contains 25 μ g/ml kantlex, again the transformant dibbling that grows in the LB flat board that contains 30 μ g/mL paraxin, obtain a positive colony, with the plasmid called after pAKC6-F19 in this positive colony.The Corynebacterium glutamicum 10147 that pAKC6 transforms in contrast.

Because the power of promotor has determined the expression amount of reporter gene, so, can infer the intensity of promotor by measuring the ratio vigor of reporter gene.Measure the total protein and the work of CAT enzyme that contain plasmid pAKC6 Corynebacterium glutamicum and contain plasmid pAKC6-F19 Corynebacterium glutamicum respectively, CAT enzyme activity determination method is as follows: the 1.0mL reaction system contains 100mmol/L Tris-HCL (PH7.8), 0.1mmol/L acetyl-CoA, 4mg/mL 5,5-two sulphur dinitrobenzoic acids (DTNB), an amount of crude enzyme liquid; Reaction mixture is heated to 37 ℃ in water-bath, adding paraxin makes and is final concentration 0.1mmol/L, and mixing is measured absorbance value A immediately ₄₁₂With the reaction solution that does not add paraxin is contrast.

The definition of CAT activity unit: a unit of activity (U) is under above-mentioned reaction conditions, the required enzyme amount of per minute acetylize 1mol paraxin.

The result shows, the Corynebacterium glutamicum that has pAKC6 can a spot of expression CAT, the CAT specific activity of enzyme is 0.21U/mg, and the CAT specific activity of enzyme that has the bacterial strain of pAKC6-F19 is 25.15U/mg, be the former 120 times, illustrate that the F19 fragment has promoter function, promotor carrier detection pAKC6 can be used for screening the fragment with promoter function.

Further this promoter fragment is checked order, the segmental length of F19 is 632bp.Through the BLASTN comparison, show that the Strain-specific DNA island 9 of F19 and Corynebacterium glutamicum R strain has 99% homology.Use BDGP Neural Network Promoter Prediction V2.2 that F19 is analyzed, find one section and must be divided into 0.97 promotor,-10 districts of this promotor potential-10 district (TGGGGT) and-35 districts (TTTACT) and standard and the similarity in-35 districts are lower, but the 18bp of the standard that is spaced apart of the two, and these-10 districts (TGGGGT) are in full accord with-10 districts (TGGGGT) of the Corynebacterium glutamicum promotor P34 that has reported, show that it should be able to be the identification of Corynebacterium glutamicum RNA polymerase.The segmental promoter region sequence of this F19 is as shown in table 4.

The sequence of table 4. promotor

Sequence table

＜110〉Institute of Microorganism, Academia Sinica

＜120〉a kind of shuttle plasmid and the plasmid of deriving thereof

<130>CGGNARW81156

<160>2

<210>1

<211>5684

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

ggtaccgagc?tcgaattcag?atctaagctt?ctgcagtcta?gagcatgcag?cttggcgtaa 60

tcatggtcat?agctgtttcc?tgtgtgaaat?tgttatccgc?tcacaattcc?acacaacata 120

cgagccggaa?gcataaagtg?taaagcctgg?ggtgcctaat?gagtgagcta?actcacatta 180

attgcgttgc?gctcactgcc?cgctttccag?tcgggaaacc?tgtcgtgcca?gctgcattaa 240

tgaatcggcc?aacgcgcggg?gagaggcggt?ttgcgtattg?ggcgctcttc?cgcttcctcg 300

ctcactgact?cgctgcgctc?ggtcgttcgg?ctgcggcgag?cggtatcagc?tcactcaaag 360

gcggtaatac?ggttatccac?agaatcaggg?gataacgcag?gaaagaacat?gtgagcaaaa 420

ggccagcaaa?aggccaggaa?ccgtaaaaag?gccgcgttgc?tggcgttttt?ccataggctc 480

cgcccccctg?acgagcatca?caaaaatcga?cgctcaagtc?agaggtggcg?aaacccgaca 540

ggactataaa?gataccaggc?gtttccccct?ggaagctccc?tcgtgcgctc?tcctgttccg 600

accctgccgc?ttaccggata?cctgtccgcc?tttctccctt?cgggaagcgt?ggcgctttct 660

catagctcac?gctgtaggta?tctcagttcg?gtgtaggtcg?ttcgctccaa?gctgggctgt 720

gtgcacgaac?cccccgttca?gcccgaccgc?tgcgccttat?ccggtaacta?tcgtcttgag 780

tccaacccgg?taagacacga?cttatcgcca?ctggcagcag?ccactggtaa?caggattagc 840

agagcgaggt?atgtaggcgg?tgctacagag?ttcttgaagt?ggtggcctaa?ctacggctac 900

actagaagaa?cagtatttgg?tatctgcgct?ctgctgaagc?cagttacctt?cggaaaaaga 960

gttggtagct?cttgatccgg?caaacaaacc?accgctggta?gcggtggttt?ttttgtttgc 1020

aagcagcaga?ttacgcgcag?aaaaaaagga?tctcaagaag?atcctttgat?cttttctacg 1080

gggtctgacg?ctcagtggaa?cgaaaactca?cgttaaggga?ttttggtcat?gagattatca 1140

aaaaggatct?tcacctagat?ccttttaaat?taaaaatgaa?gttttaaatc?aatctaaagt 1200

atatatgagt?aaacttggtc?tgacagttac?caatgcttaa?tcagtgaggc?acctatctca 1260

gcgatctgtc?tatttcgttc?atccatagtt?gcctgactcc?ccgtcgtgta?gataactacg 1320

atacgggagg?gcttaccatc?tggccccagt?gctgcaatga?taccgcgaga?cccacgctca 1380

ccggctccag?atttatcagc?aataaaccag?ccagccggaa?gggccgagcg?cagaagtggt 1440

cctgcaactt?tatccgcctc?catccagtct?attaattgtt?gccgggaagc?tagagtaagt 1500

agttcgccag?ttaatagttt?gcgcaacgtt?gttgccattg?ctacaggcat?cgtggtgtca 1560

cgctcgtcgt?ttggtatggc?ttcattcagc?tccggttccc?aacgatcaag?gcgagttaca 1620

tgatccccca?tgttgtgcaa?aaaagcggtt?agctccttcg?gtcctccgat?cgttgtcaga 1680

agtaagttgg?ccgcagtgtt?atcactcatg?gttatggcag?cactgcataa?ttctcttact 1740

gtcatgccat?ccgtaagatg?cttttctgtg?actggtgagt?actcaaccaa?gtcattctga 1800

gaatagtgta?tgcggcgacc?gagttgctct?tgcccggcgt?caatacggga?taataccgcg 1860

ccacatagca?gaactttaaa?agtgctcatc?attggaaaac?gttcttcggg?gcgaaaactc 1920

tcaaggatct?taccgctgtt?gagatccagt?tcgatgtaac?ccactcgtgc?acccaactga 1980

tcttcagcat?cttttacttt?caccagcgtt?tctgggtgag?caaaaacagg?aaggcaaaat 2040

gccgcaaaaa?agggaataag?ggcgacacgg?aaatgttgaa?tactcatact?cttccttttt 2100

caatattatt?gaagcattta?tcagggttat?tgtctcatga?gcggatacat?atttgaatgt 2160

atttagaaaa?ataaacaaat?aggggttccg?cgcacatttc?cccgaaaagt?gccacctgac 2220

gtcgactgtt?cacgcggtcc?tcgtccgttg?ccgcgtgaac?cgcctctcgc?atatcgatcg 2280

tgtcactggc?gagccattgc?ggcgatatga?gcatcctcat?ccgggatcgt?tgattcatgt 2340

cgatgtcacg?aagttcggca?acatccccga?cggcggtgga?catcgttacg?taggtcggca 2400

gcaaggcgca?cggaacaagc?tagcgactcc?gggattacca?cgaggaaaag?atcacaagcc 2460

gcgcaccggg?acggcgttcg?ttcacacagt?catcgatgac?cactcccgcg?tcgcatacgc 2520

agaaatctgg?tcggatgagc?aggcgagcac?agcggtggga?gttctcgaac?gcgccgtggc 2580

ctggttcgcc?gaacgaggcg?tgaccgtcga?gcgagtccta?tccgacaacg?ggtcggcata 2640

cagatcccac?gcatggaggg?acttctgcgc?tcggctcggc?atccgacaca?agcggacacg 2700

ccctaccggc?cgcagacgaa?cgggaagatc?gagcgattcc?acgcacgctc?ggggacggct 2760

gggcctatgc?caggttttac?ggttcagagg?ccgaacgacg?ctggcgctgc?ccggctggct 2820

ccacttctac?aaccaccacc?gacaccactc?tgcgattggc?ggcgtaccct?tcgaccgact 2880

caacaacgtc?cctggacatc?acatctagcc?caagcgcgat?gagcaccagg?gcgccgatga 2940

cggcgagcag?aagtagtgcg?aggggattgc?gttgaaaaag?tttcacgggg?ttctccttgt 3000

gttgcgtttg?attgtctcgg?ctacaccgtg?cggtcagaac?cgcgttgtgc?aaccagatgg 3060

caaatatcag?ttccaagaaa?tttcgagccg?tgaaaaaagt?gtctcagtga?ggtgtgcacg 3120

ttttcggagc?caaggcagac?gcgcagcggg?tgccgcggca?gcccctagaa?cgcccgtagg 3180

agccaccagg?aggcttgagc?caacctcagg?gagggggtag?agccctgaga?gccttagagg 3240

gctacctgag?ggcgccggcc?aaaggcacgg?ttcgctcgca?ccggaagggt?tcttcagggg 3300

aaccccctga?aaccccggaa?acatctgact?tggttacgat?ggactttgaa?cacgccgagg 3360

aatgaacccc?gaccgcgcac?agtcatatga?gaaagtcagc?tgcatgagac?catccacccg 3420

ccctgaggga?cgctttgagc?agctctggct?accgctatgg?ccactagcaa?gcgacaacct 3480

ccgtgacggc?atctaccgca?cctcacgcga?taaagcccta?gacaagcgct?atatcgaaac 3540

caacccggat?gccatctcga?atctcctggt?cgtcgacatc?gacaagcaag?acgcactctt 3600

gcgttcgatg?tgggacagag?acggctggcg?gcctaacgcc?gtggttgaaa?acccttaaac 3660

gggcacgcac?acgccgtgtg?ggcgctcgcg?gagccattta?cccgcaccga?gtacgccagg 3720

cgcaagccac?ctgctacgcg?gccgctgtca?ccgaaggact?acgccgctct?gtcgacggcg 3780

acaaaggcta?ctccgggctg?atcaccaaaa?accccgagca?cacagcctgg?gacagccact 3840

gggtaaccga?taagctctac?acactcgatg?aactgcgctt?ttggctggaa?gaaaccggct 3900

tcatgccgcc?agagtcctgg?aagaaaaccc?gcagaaaatc?gcccatcggg?ctaggccgaa 3960

actgcgcgct?cttcgagagc?gcacgcagct?gggcttatcg?cgaaatacgc?caccacttcg 4020

gcgaccccga?cgggctagga?cgctctatcc?aagccaccgc?ccaggcgctc?aaccaggagc 4080

tgttttctga?gccgctaccc?gtggcagaag?ttgaccaaat?agccaggtca?atccaccgat 4140

ggatcatcac?caaatcacgc?atgtggacag?acggcccagc?cgtctacgaa?gccacattca 4200

ccacaatcca?agccacacgc?ggaaaacgaa?gcgccgagca?ccgctggggc?accaccaacg 4260

cggaacgaat?cgaaagattt?atcaatgact?aagcgcactc?gtattccccg?aaacggtaaa 4320

actatccgcg?aagtagccga?aggaactggc?ctatcaactg?cgacaatcga?acgctggaca 4380

tctgcccctc?gcgaagatta?tctagcgcag?gccaatgaaa?agcgtgtccg?cgtccaggaa 4440

ctgcgtgcca?aaggtctgtc?catgcgcgct?atcgcggcgg?aaatcggttg?ctcagtcggc 4500

ctggttcatc?gctacgtcaa?agaagttgaa?gagaagaaaa?ccgcgtaaat?ccagcggttt 4560

agtccccctc?ggcgtgttca?taataggtca?cgaaccaagt?cagatatttc?cggggtttag 4620

cgcgtctttt?gctggcgtgc?gagcttgcgg?cgttgctgtc?gagtgagcgt?ttgttgggct 4680

ggttggtcta?cgacagcgac?tagaggatcc?cctggatacc?gctcgccgca?gccgaacgac 4740

cgagcgcagc?gagtcagtga?gcgaggaagc?ggaagaatca?aattatcgag?gttgacacct 4800

tttgccgatt?ctggtagaat?gacaccaaca?tagatcgatc?tgatcaagag?acaggatgag 4860

gatcgtttcg?catgattgaa?caagatggat?tgcacgcagg?ttctccggcc?gcttgggtgg 4920

agaggctatt?cggctatgac?tgggcacaac?agacaatcgg?ctgctctgat?gccgccgtgt 4980

tccggctgtc?agcgcagggg?cgcccggttc?tttttgtcaa?gaccgacctg?tccggtgccc 5040

tgaatgaact?gcaagacgag?gcagcgcggc?tatcgtggct?ggccacgacg?ggcgttcctt 5100

gcgcagctgt?gctcgacgtt?gtcactgaag?cgggaaggga?ctggctgcta?ttgggcgaag 5160

tgccggggca?ggatctcctg?tcatctcacc?ttgctcctgc?cgagaaagta?tccatcatgg 5220

ctgatgcaat?gcggcggctg?catacgcttg?atccggctac?ctgcccattc?gaccaccaag 5280

cgaaacatcg?catcgagcga?gcacgtactc?ggatggaagc?cggtcttgtc?gatcaggatg 5340

atctggacga?agagcatcag?gggctcgcgc?cagccgaact?gttcgccagg?ctcaaggcgc 5400

acatacccga?cggcgaggat?ctcgtcgtga?cccatggcga?tgcctgcttg?ccgaatatca 5460

tggtggaaaa?tggccgcttt?tctggattca?tcgactgtgg?ccggctgggt?gtggcggacc 5520

gctatcagga?catagcgttg?gctacccgtg?atattgctga?agagcttggc?ggcgaatggg 5580

ctgaccgctt?cctcgtgctt?tacggtatcg?ccgctcccga?ttcgcagcgc?atcgccttct 5640

atcgccttct?tgacgagttc?ttctgagcgg?gactctgggg?ttcg 5684

<210>2

<211>6474

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

ggtacccctc?gccttttatg?gcgaggggtt?tactttgaat?tcagatctaa?gcttaagtaa 60

ttaacaggag?ctaaggaagc?taaaatggag?aaaaaaatca?ctggatatac?caccgttgat 120

atatcccaat?ggcatcgtaa?agaacatttt?gaggcatttc?agtcagttgc?tcaatgtacc 180

tataaccaga?ccgttcagct?ggatattacg?gcctttttaa?agaccgtaaa?gaaaaataag 240

cacaagtttt?atccggcctt?tattcacatt?cttgcccgcc?tgatgaatgc?tcatccggaa 300

ttccgtatgg?caatgaaaga?cggtgagctg?gtgatatggg?atagtgttca?cccttgttac 360

accgttttcc?atgagcaaac?tgaaacgttt?tcatcgctct?ggagtgaata?ccacgacgat 420

ttccggcagt?ttctacacat?atattcgcaa?gatgtggcgt?gttacggtga?aaacctggcc 480

tatttcccta?aagggtttat?tgagaatatg?tttttcgtct?cagccaatcc?ctgggtgagt 540

ttcaccagtt?ttgatttaaa?cgtggccaat?atggacaact?tcttcgcccc?cgttttcacc 600

atgggcaaat?attatacgca?aggcgacaag?gtgctgatgc?cgctggcgat?tcaggttcat 660

catgccgtct?gtgatggctt?ccatgtcggc?agaatgctta?atgaattaca?acagtactgc 720

gatgagtggc?agggcggggc?gtaatttttt?taaggcagtt?attggtgccc?ttaaacgcct 780

ggtgctacgc?ctgaataagt?gataataagc?ggatgaatgg?cagaaatcta?gagcatgcag 840

cttggcgtaa?tcatggtcat?agctgtttcc?tgtgtgaaat?tgttatccgc?tcacaattcc 900

acacaacata?cgagccggaa?gcataaagtg?taaagcctgg?ggtgcctaat?gagtgagcta 960

actcacatta?attgcgttgc?gctcactgcc?cgctttccag?tcgggaaacc?tgtcgtgcca 1020

gctgcattaa?tgaatcggcc?aacgcgcggg?gagaggcggt?ttgcgtattg?ggcgctcttc 1080

cgcttcctcg?ctcactgact?cgctgcgctc?ggtcgttcgg?ctgcggcgag?cggtatcagc 1140

tcactcaaag?gcggtaatac?ggttatccac?agaatcaggg?gataacgcag?gaaagaacat 1200

gtgagcaaaa?ggccagcaaa?aggccaggaa?ccgtaaaaag?gccgcgttgc?tggcgttttt 1260

ccataggctc?cgcccccctg?acgagcatca?caaaaatcga?cgctcaagtc?agaggtggcg 1320

aaacccgaca?ggactataaa?gataccaggc?gtttccccct?ggaagctccc?tcgtgcgctc 1380

tcctgttccg?accctgccgc?ttaccggata?cctgtccgcc?tttctccctt?cgggaagcgt 1440

ggcgctttct?catagctcac?gctgtaggta?tctcagttcg?gtgtaggtcg?ttcgctccaa 1500

gctgggctgt?gtgcacgaac?cccccgttca?gcccgaccgc?tgcgccttat?ccggtaacta 1560

tcgtcttgag?tccaacccgg?taagacacga?cttatcgcca?ctggcagcag?ccactggtaa 1620

caggattagc?agagcgaggt?atgtaggcgg?tgctacagag?ttcttgaagt?ggtggcctaa 1680

ctacggctac?actagaagaa?cagtatttgg?tatctgcgct?ctgctgaagc?cagttacctt 1740

cggaaaaaga?gttggtagct?cttgatccgg?caaacaaacc?accgctggta?gcggtggttt 1800

ttttgtttgc?aagcagcaga?ttacgcgcag?aaaaaaagga?tctcaagaag?atcctttgat 1860

cttttctacg?gggtctgacg?ctcagtggaa?cgaaaactca?cgttaaggga?ttttggtcat 1920

gagattatca?aaaaggatct?tcacctagat?ccttttaaat?taaaaatgaa?gttttaaatc 1980

aatctaaagt?atatatgagt?aaacttggtc?tgacagttac?caatgcttaa?tcagtgaggc 2040

acctatctca?gcgatctgtc?tatttcgttc?atccatagtt?gcctgactcc?ccgtcgtgta 2100

gataactacg?atacgggagg?gcttaccatc?tggccccagt?gctgcaatga?taccgcgaga 2160

cccacgctca?ccggctccag?atttatcagc?aataaaccag?ccagccggaa?gggccgagcg 2220

cagaagtggt?cctgcaactt?tatccgcctc?catccagtct?attaattgtt?gccgggaagc 2280

tagagtaagt?agttcgccag?ttaatagttt?gcgcaacgtt?gttgccattg?ctacaggcat 2340

cgtggtgtca?cgctcgtcgt?ttggtatggc?ttcattcagc?tccggttccc?aacgatcaag 2400

gcgagttaca?tgatccccca?tgttgtgcaa?aaaagcggtt?agctccttcg?gtcctccgat 2460

cgttgtcaga?agtaagttgg?ccgcagtgtt?atcactcatg?gttatggcag?cactgcataa 2520

ttctcttact?gtcatgccat?ccgtaagatg?cttttctgtg?actggtgagt?actcaaccaa 2580

gtcattctga?gaatagtgta?tgcggcgacc?gagttgctct?tgcccggcgt?caatacggga 2640

taataccgcg?ccacatagca?gaactttaaa?agtgctcatc?attggaaaac?gttcttcggg 2700

gcgaaaactc?tcaaggatct?taccgctgtt?gagatccagt?tcgatgtaac?ccactcgtgc 2760

acccaactga?tcttcagcat?cttttacttt?caccagcgtt?tctgggtgag?caaaaacagg 2820

aaggcaaaat?gccgcaaaaa?agggaataag?ggcgacacgg?aaatgttgaa?tactcatact 2880

cttccttttt?caatattatt?gaagcattta?tcagggttat?tgtctcatga?gcggatacat 2940

atttgaatgt?atttagaaaa?ataaacaaat?aggggttccg?cgcacatttc?cccgaaaagt 3000

gccacctgac?gtcgactgtt?cacgcggtcc?tcgtccgttg?ccgcgtgaac?cgcctctcgc 3060

atatcgatcg?tgtcactggc?gagccattgc?ggcgatatga?gcatcctcat?ccgggatcgt 3120

tgattcatgt?cgatgtcacg?aagttcggca?acatccccga?cggcggtgga?catcgttacg 3180

taggtcggca?gcaaggcgca?cggaacaagc?tagcgactcc?gggattacca?cgaggaaaag 3240

atcacaagcc?gcgcaccggg?acggcgttcg?ttcacacagt?catcgatgac?cactcccgcg 3300

tcgcatacgc?agaaatctgg?tcggatgagc?aggcgagcac?agcggtggga?gttctcgaac 3360

gcgccgtggc?ctggttcgcc?gaacgaggcg?tgaccgtcga?gcgagtccta?tccgacaacg 3420

ggtcggcata?cagatcccac?gcatggaggg?acttctgcgc?tcggctcggc?atccgacaca 3480

agcggacacg?ccctaccggc?cgcagacgaa?cgggaagatc?gagcgattcc?acgcacgctc 3540

ggggacggct?gggcctatgc?caggttttac?ggttcagagg?ccgaacgacg?ctggcgctgc 3600

ccggctggct?ccacttctac?aaccaccacc?gacaccactc?tgcgattggc?ggcgtaccct 3660

tcgaccgact?caacaacgtc?cctggacatc?acatctagcc?caagcgcgat?gagcaccagg 3720

gcgccgatga?cggcgagcag?aagtagtgcg?aggggattgc?gttgaaaaag?tttcacgggg 3780

ttctccttgt?gttgcgtttg?attgtctcgg?ctacaccgtg?cggtcagaac?cgcgttgtgc 3840

aaccagatgg?caaatatcag?ttccaagaaa?tttcgagccg?tgaaaaaagt?gtctcagtga 3900

ggtgtgcacg?ttttcggagc?caaggcagac?gcgcagcggg?tgccgcggca?gcccctagaa 3960

cgcccgtagg?agccaccagg?aggcttgagc?caacctcagg?gagggggtag?agccctgaga 4020

gccttagagg?gctacctgag?ggcgccggcc?aaaggcacgg?ttcgctcgca?ccggaagggt 4080

tcttcagggg?aaccccctga?aaccccggaa?acatctgact?tggttacgat?ggactttgaa 4140

cacgccgagg?aatgaacccc?gaccgcgcac?agtcatatga?gaaagtcagc?tgcatgagac 4200

catccacccg?ccctgaggga?cgctttgagc?agctctggct?accgctatgg?ccactagcaa 4260

gcgacaacct?ccgtgacggc?atctaccgca?cctcacgcga?taaagcccta?gacaagcgct 4320

atatcgaaac?caacccggat?gccatctcga?atctcctggt?cgtcgacatc?gacaagcaag 4380

acgcactctt?gcgttcgatg?tgggacagag?acggctggcg?gcctaacgcc?gtggttgaaa 4440

acccttaaac?gggcacgcac?acgccgtgtg?ggcgctcgcg?gagccattta?cccgcaccga 4500

gtacgccagg?cgcaagccac?ctgctacgcg?gccgctgtca?ccgaaggact?acgccgctct 4560

gtcgacggcg?acaaaggcta?ctccgggctg?atcaccaaaa?accccgagca?cacagcctgg 4620

gacagccact?gggtaaccga?taagctctac?acactcgatg?aactgcgctt?ttggctggaa 4680

gaaaccggct?tcatgccgcc?agagtcctgg?aagaaaaccc?gcagaaaatc?gcccatcggg 4740

ctaggccgaa?actgcgcgct?cttcgagagc?gcacgcagct?gggcttatcg?cgaaatacgc 4800

caccacttcg?gcgaccccga?cgggctagga?cgctctatcc?aagccaccgc?ccaggcgctc 4860

aaccaggagc?tgttttctga?gccgctaccc?gtggcagaag?ttgaccaaat?agccaggtca 4920

atccaccgat?ggatcatcac?caaatcacgc?atgtggacag?acggcccagc?cgtctacgaa 4980

gccacattca?ccacaatcca?agccacacgc?ggaaaacgaa?gcgccgagca?ccgctggggc 5040

accaccaacg?cggaacgaat?cgaaagattt?atcaatgact?aagcgcactc?gtattccccg 5100

aaacggtaaa?actatccgcg?aagtagccga?aggaactggc?ctatcaactg?cgacaatcga 5160

acgctggaca?tctgcccctc?gcgaagatta?tctagcgcag?gccaatgaaa?agcgtgtccg 5220

cgtccaggaa?ctgcgtgcca?aaggtctgtc?catgcgcgct?atcgcggcgg?aaatcggttg 5280

ctcagtcggc?ctggttcatc?gctacgtcaa?agaagttgaa?gagaagaaaa?ccgcgtaaat 5340

ccagcggttt?agtccccctc?ggcgtgttca?taataggtca?cgaaccaagt?cagatatttc 5400

cggggtttag?cgcgtctttt?gctggcgtgc?gagcttgcgg?cgttgctgtc?gagtgagcgt 5460

ttgttgggct?ggttggtcta?cgacagcgac?tagaggatcc?cctggatacc?gctcgccgca 5520

gccgaacgac?cgagcgcagc?gagtcagtga?gcgaggaagc?ggaagaatca?aattatcgag 5580

gttgacacct?tttgccgatt?ctggtagaat?gacaccaaca?tagatcgatc?tgatcaagag 5640

acaggatgag?gatcgtttcg?catgattgaa?caagatggat?tgcacgcagg?ttctccggcc 5700

gcttgggtgg?agaggctatt?cggctatgac?tgggcacaac?agacaatcgg?ctgctctgat 5760

gccgccgtgt?tccggctgtc?agcgcagggg?cgcccggttc?tttttgtcaa?gaccgacctg 5820

tccggtgccc?tgaatgaact?gcaagacgag?gcagcgcggc?tatcgtggct?ggccacgacg 5880

ggcgttcctt?gcgcagctgt?gctcgacgtt?gtcactgaag?cgggaaggga?ctggctgcta 5940

ttgggcgaag?tgccggggca?ggatctcctg?tcatctcacc?ttgctcctgc?cgagaaagta 6000

tccatcatgg?ctgatgcaat?gcggcggctg?catacgcttg?atccggctac?ctgcccattc 6060

gaccaccaag?cgaaacatcg?catcgagcga?gcacgtactc?ggatggaagc?cggtcttgtc 6120

gatcaggatg?atctggacga?agagcatcag?gggctcgcgc?cagccgaact?gttcgccagg 6180

ctcaaggcgc?acatacccga?cggcgaggat?ctcgtcgtga?cccatggcga?tgcctgcttg 6240

ccgaatatca?tggtggaaaa?tggccgcttt?tctggattca?tcgactgtgg?ccggctgggt 6300

gtggcggacc?gctatcagga?catagcgttg?gctacccgtg?atattgctga?agagcttggc 6360

ggcgaatggg?ctgaccgctt?cctcgtgctt?tacggtatcg?ccgctcccga?ttcgcagcgc 6420

atcgccttct?atcgccttct?tgacgagttc?ttctgagcgg?gactctgggg?ttcg 6474

Claims (6)

1. shuttle plasmid, its nucleotide sequence are in the sequence table shown in the sequence 1.

2. plasmid according to claim 1 is characterized in that: sequence 1 is kalamycin resistance gene and promotor thereof from 5 ' terminal 4872-5664 position deoxyribonucleotide in the described sequence table.

3. shuttle plasmid, be with sequence in the sequence table 1 after the kalamycin resistance gene of 5 ' terminal 4872-5664 position and promotor thereof are carried out inversion, insert in its multiple clone site that reporter gene and Corynebacterium glutamicum LeuB gene transcription terminator obtain; The direction of described reporter gene is opposite with described kalamycin resistance gene and promotor direction thereof; Described Corynebacterium glutamicum LeuB gene transcription terminator is between described reporter gene and described kalamycin resistance gene;

Described reporter gene is a chloramphenicol acetyl transferasegene; The nucleotide sequence of described chloramphenicol acetyl transferasegene be in the sequence table sequence 2 from shown in the deoxyribonucleotide of 5 ' terminal 50-827 position.

4. plasmid according to claim 3 is characterized in that: the nucleotide sequence of described Corynebacterium glutamicum LeuB gene transcription terminator be in the sequence table sequence 2 from shown in the deoxyribonucleotide of 5 ' terminal 6-37 position.

5. plasmid according to claim 4 is characterized in that: the nucleotide sequence of described plasmid is the sequence 2 in the sequence table.

6. the application of the arbitrary described plasmid of claim 1-5 in genetically engineered.

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