CN103451181B - A kind of resistance expression's box for efficiently building non-resistant mark recombinant mycobacterium - Google Patents

Abstract

The invention discloses a resistance expression cassette for efficiently constructing recombinant mycobacteria without resistance markers. The resistance expression cassette contains a simplified hygromycin resistance gene (shown in SEQ ID NO: 1), and dif1 and dif2 sequences located at both ends of the hygromycin resistance gene. The simplified Hyg gene sequence of the present invention is shortened from the original 1.7kb to about 1kb, and complex structures such as transcription terminators and longer natural promoters are removed, which is convenient for PCR amplification, and there are fewer internal enzyme cutting sites (common enzyme cutting site has been site-directed mutation), and at the same time, Hyg comes with a short artificial promoter, which can be expressed in both Escherichia coli and mycobacteria, and the transcription and translation of downstream genes will not be affected when directional insertion. The resistance expression cassette of the present invention adds a dif sequence to both ends of the simplified Hyg gene, which can automatically dissociate in mycobacteria. After the Hyg gene is lost, the damaged gene can still continue to express the shortened small peptide without affecting the translation of downstream genes, thus effectively avoiding the polarity effect.

Description

A resistance expression cassette for efficiently constructing recombinant mycobacteria without resistance markers

technical field

The invention belongs to the field of genetic engineering, and in particular relates to a resistance expression cassette for efficiently constructing recombinant mycobacteria without resistance markers and an application thereof.

Background technique

Mtb is the pathogenic bacterium that causes tuberculosis, which can invade various organs of the body, but tuberculosis is the most common. Tuberculosis is still an important infectious disease, and its diagnosis, prevention and treatment are progressing slowly. These three aspects are all related to the study of the function of Mtb gene, and the genetic modification of Mtb plays a pivotal role in the study of Mtb. Mycobacterium tuberculosis grows slowly, it is difficult to carry out genetic manipulation on it, and requires an expensive level 3 biosafety laboratory with negative pressure. Long-term cultivation not only takes up space but is easy to contaminate, etc., making the research on Mycobacterium tuberculosis time-consuming and expensive exhausting. Taking the research and development of anti-Mtb drugs as an example, the research and development of anti-Mtb drugs is expensive and takes a long time. For nearly half a century, until the end of 2012, a drug with a new mechanism of action has just come out, and it has potential toxicity. One of the core issues in the current research on anti-Mtb drugs/vaccine is to establish effective, fast and cheap genetically engineered bacteria.

Currently, only the kanamycin resistance gene and the hygromycin resistance gene are relatively effective in the genetic manipulation of mycobacteria. Therefore, when conducting research on mycobacterial mutant strains, if the strains used have resistance genes, this will bring inconvenience to future genetic manipulations. For example, gene knockout/genetic complementation requires at least two resistance genes; genetic recombination technology (recombineering) also requires two resistance genes, etc. At the same time, in mycobacteria, it is sometimes necessary to express multiple antigenic proteins and multiple genetic manipulations. Therefore, the generation of non-resistant recombinant strains is particularly important for subsequent manipulations. Gene knockout technology can be used to replace the target gene with a resistance gene, but phage packaging technology is often used for gene knockout. This technology needs to package the upstream sequence of the target gene + the resistance gene + the downstream sequence of the target gene (referred to as the three fragments) together into the phage to form a phagemid. The total length of the three fragments has a certain limit. If it is too long, it will not be packaged into phages, and phagemids for gene knockout cannot be obtained. Due to the limited capacity of the packaging, if the resistance gene is short, the length of the upstream sequence of the target gene + the downstream sequence of the target gene can be appropriately increased, and other elements can even be added. Therefore, it is beneficial to appropriately increase the length of the effective DNA fragment for phage packaging. After gene knockout, one of the most effective detection methods is PCR verification. However, the transcription terminator of the previous Hyg gene has a complex secondary structure, making PCR difficult. Some inventions add Res and other sequences to both sides of Hyg , and Res and other sequences need to artificially express a foreign protein to recognize and act on Hyg before it can be dissociated. That is, in actual operation, the plasmid with the Res-Hyg-Res sequence is first introduced into mycobacteria, and then the mycobacteria after the Res-Hyg-Res sequence is inserted into the genome are screened, and then another plasmid that can express the dissociated protein is introduced. , and then screened to obtain mycobacteria with Hyg loss. Many experiments often require continued screening for strains that have lost the plasmid expressing the dissociated protein. Therefore, it is very cumbersome and time-consuming. Some similar systems are extremely inefficient for resistance gene loss in mycobacteria. The addition of dif sequence to both sides of Hyg facilitates the dissociation of Hyg gene, which has only been reported in recent years. The advantage is that there is no need to artificially express foreign proteins to realize the dissociation of resistance genes, because the mycobacterium itself encodes such proteins, thus saving a lot of unnecessary troubles. Moreover, the efficiency of the system is very high, and the compatibility is good (for example, the dif sequence of the slow-growing Mycobacterium tuberculosis also works in the fast-growing Mycobacterium smegmatis). However, in the research using the dif sequence system, the Hyg resistance gene used is a long fragment (1.7kb) with a complex secondary structure at the 3' end. There are common enzyme cutting sites inside, and both ends can be cut by enzymes There are few sites, and the constructed resistance expression cassette cannot be "universal".

Therefore, if it is possible to construct a shorter, genetically manipulated universal resistance expression cassette with multiple cloning sites with automatic dissociation function in mycobacteria, and without polarity effects, it will greatly simplify Genetic manipulation of diverse mycobacteria. During the genetic manipulation of mycobacteria, because the resistance gene introduced by the resistance expression cassette can be dissociated automatically, the subsequent genetic manipulation will not be limited by the selection of the resistance gene, and there is no resistance gene The mutant strains also make a variety of genetic studies more possible, and will provide a powerful tool for the genetic engineering research of mycobacteria.

Contents of the invention

One object of the present invention is to provide a simplified hygromycin resistance gene Hyg with its own artificial promoter.

Another object of the present invention is to provide a resistance expression cassette for efficiently constructing recombinant mycobacteria without resistance markers.

Another object of the present invention is to provide a recombinant plasmid for efficiently constructing recombinant mycobacteria without resistance markers.

The technical scheme that the present invention takes is:

A hygromycin resistance gene after simplification and transformation, its nucleotide sequence is shown in SEQ ID NO:1.

A resistance expression cassette for efficiently constructing recombinant mycobacteria without resistance markers, which contains a simplified and transformed hygromycin resistance gene, and dif1 and dif2 sequences located at both ends of the hygromycin resistance gene, its characteristics In that, the nucleotide sequence of the simplified and modified hygromycin resistance gene is shown in SEQ ID NO: 1, and the sequences of dif1 and dif2 are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The resistance expression cassette is named "dif- ΩHYG - dif resistance expression cassette" here .

Preferably, multiple enzyme cutting sites are added to both ends of the resistance expression cassette.

The nucleotide sequence of the resistance expression cassette is shown in SEQ ID NO:5 or SEQ ID NO:6.

Recombinant plasmid containing the dif- ΩHYG - dif resistance expression cassette described above. Preferably, the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO:2.

A recombinant plasmid for efficiently constructing recombinant mycobacteria without resistance markers, which contains: a promoter, a phage integration site, an integrase gene, a dif- ΩHYG - dif resistance expression box, and a replication origin site.

In a clockwise direction, the phage integration site, integrase gene, and dif- ΩHYG - dif resistance expression cassette are sequentially linked together.

The promoter is a LacZ promoter or a BLA promoter, and the replication initiation site is an Escherichia coli replication origin.

Preferably, the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO:4.

The beneficial effects of the present invention are:

(1) The simplified Hyg gene sequence of the present invention is shortened from the original 1.7kb to about 1kb, and complex structures such as transcription terminators and longer natural promoters are removed, which is convenient for PCR amplification and has fewer internal restriction sites ( The common enzyme cutting site has been mutated), meanwhile, Hyg has a short artificial promoter, which can be expressed in both Escherichia coli and mycobacteria, and the transcription and translation of downstream genes will not be affected by directional insertion.

(2) The resistance expression cassette of the present invention adds a dif sequence at both ends of the simplified Hyg gene, which can automatically dissociate in mycobacteria. After the Hyg gene is lost, the damaged gene can still continue to express the shortened small peptide without affecting the translation of downstream genes, thus effectively avoiding the polarity effect.

(3) Multiple enzyme cutting sites are added to both ends of the resistance expression cassette of the present invention, so that the expression cassette can be inserted into a specific sequence, and it is also convenient to add other sequences on both sides.

Description of drawings

Figure 1 is a schematic diagram of the structure of the plasmid pUCDHmKE;

Figure 2 is a map of the restriction sites of plasmid pUCDHmKE;

Fig. 3 is the construction flowchart of plasmid pUCDHmKE;

Figure 4 is a schematic diagram of the structure of the integrated plasmid pMH94DHmKE;

Figure 5 is a flow chart of the construction of the integrated plasmid pMH94DHmKE;

Figure 6 is a schematic diagram of the structure of the integrated plasmid pblDHCiGn;

Fig. 7 is the construction flowchart of plasmid pblDHCiGn;

Figure 8 is a schematic diagram comparing the dif -ΩHYG- dif expression cassette with the fragment containing the hygromycin (HYG) resistance gene ( Hyg ) commonly used in the genetic manipulation of mycobacteria in the past (P1, P2, P3: putative Hyg Promoter; Pa: putative Hyg artificial promoter, which covers dif1 in the dif - ΩHYG - dif expression cassette, commonly used restriction sites: E1, EcoRI (removed in our dif -ΩHYG- dif expression cassette out), Nt, Not I; Sp, Spe I; B, BamH I, E5, Eco RV; Nd, Nde I; H, Hin dIII; Xo, Xho I; C, Cla I; Xb, Xba I; S, Sma I; Nc, Nco I; P, Pst I; Nh, Nhe I; K, Kpn I);

Figure 9 is the analysis of the sequence of Hin dIII- Xho I- dif - Xho I- Hin dIII present in the pUCDHmke plasmid in the dif positive chain (A) and the negative chain (B) and the corresponding expressed polypeptide (they are dif - ΩHYG - the sequence remaining in the genome after dissociation of the expression cassette V1 of the dif. Spe I- Bam HI- Eco RV- Eco RI- Nde I- Hin dIII- Xho I- dif - Xho I- Hin dIII- Nco I- Pst I- Analysis of the sequence of Nhe I (present in pblDHC1n) in the negative strand of dif and its corresponding expressed polypeptide (C), which is the sequence left in the genome after dissociation of the dif- ΩHYG - dif expression cassette V2, represented by The amino acids encoded by the three reading frames are indicated by blue capital letters, the underlined part indicates the amino acids encoded by the dif sequence in the open reading frame, and the asterisk (*) indicates the terminator).

Detailed ways

The present invention will be further described below in conjunction with the examples, but not limited thereto.

The molecular biology experimental techniques used in the following examples include PCR amplification, plasmid extraction, plasmid transformation, DNA fragment ligation, enzyme digestion, gel electrophoresis, etc., unless otherwise specified, generally operate according to conventional methods. For details, please refer to " Molecular Cloning Experiment Guide (Third Edition) (translated by Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitang, etc., 2002, Beijing: Science Press), or according to the conditions suggested by the manufacturer.

The pfu enzymes, dNTPs and related reagents used in the PCR reactions in the following examples were purchased from Shanghai Sangon Biotechnology Co., Ltd.; the antibiotic hygromycin was purchased from Roche; E. coli competent DH5a was purchased from Guangzhou Dongsheng Biotechnology Co., Ltd., Cat. No. It was C1042; the DNA ligation reaction used the T4 DNA ligation kit of Takara Bao Bio Company, model: D6020A; the plasmid DNA extraction kit (BSC01M1), DNA recovery kit (BSC02M1), and PCR purification kit (BSC03M1) were purchased from Bo Nichibio Corporation.

The PCR primers and DNA fragments used in the following examples were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

Example 1 Construction of plasmid pUCDHmKE

The schematic diagram of the structure of the plasmid pUCDHmKE is shown in Figure 1, and the restriction site map is shown in Figure 2, which contains the above-mentioned dif- ΩHYG - dif resistance expression cassette. It can be seen from Figure 1 that the plasmid pUCDHmKE contains in a clockwise direction: the E. coli replication origin (pMB1 ori), the dif- ΩHYG - dif resistance expression cassette, and the promoter P (BLA) (used to initiate the ampicillin resistance gene Amp ( That is bla) , the ampicillin resistance gene Amp can be used for plasmid screening.

The dif- ΩHYG - dif resistance expression cassette contains a streamlined modified hygromycin resistance gene ( Hyg ), as described in SEQ ID NO: 1, the 24th to 73th bp of the gene is a promoter sequence (Pr ), used to initiate the expression of the Hyg gene. The dif sequences connected at both ends of the Hyg gene sequence are shown in SEQ ID NO:7 and SEQ ID NO:8.

In addition, the two ends of the dif- ΩHYG - dif resistance expression cassette are respectively connected with the Hin dIII restriction site, the Xho I restriction site, and the Xba I restriction site respectively. These three restriction sites can be used to cut out the plasmid dif- ΩHYG - dif resistance expression cassette on .

The construction flow chart of plasmid pUCDHmKE is shown in Figure 3. Plasmid pNBV1 will be used in this process (gifted by the William Bishai Laboratory of Johns Hopkins University, USA. The plasmid map is shown in Figure 3. For the specific construction method, see reference 1. In the plasmid pNBV1 Contains the original hygromycin resistance gene ( Hyg *), about 1.7kb in length.

The specific method is as follows:

1. Construction of plasmid pUCDis

Plasmid pUC19 (commercial plasmid) was digested with restriction endonucleases Kpn I and Hind III, and a 2.6kb fragment was recovered by purification and recovery kit;

Synthetic DNA fragments dif1 and dif2 (as shown in SEQ ID NO:7 and SEQ ID NO:8, synthesized by Shanghai Jierui Bioengineering Co., Ltd.), the two ends of the synthesized dif1 have Hind III and Xba I restriction sites , there are Xba I and Kpn I restriction sites at both ends of dif2 , so, connect dif1 and dif2 and the purified and recovered plasmid pUC19 (double digestion with Kpn I and Hind III) in three fragments, and transform Escherichia coli competent DH5α , use the ampicillin-resistant LB solid plate to screen out positive clones, pick a single clone and culture it in LB liquid medium, extract the plasmid, and identify the correct clone by enzyme digestion, which is the plasmid pUCDis.

2. Construction of plasmid pUCDHmKE

Amplified fragment Hyg : In this step, plasmid pNBV1 is used as a template, and primers Hygf2 (5'-TG TCTAGACCGGCCGTGCGGAATTAA-3') (SEQ ID NO:9) and Hygr727 (5'-ATTCTAGATCAGGCGCCGGGGGCGGTGTC-3') (SEQ ID NO:10 ) for amplification. This pair of primers can amplify a Hyg fragment of about 1.1kb from the plasmid pNBV1, which is significantly more streamlined than the original 1.7kb, and the streamlined Hyg gene can still express and function as a resistance gene, and its efficiency is the same as that in the original plasmid. The Hyg * genes expressed resistance with comparable efficiency.

Both ends of the amplified fragment have Xba I restriction sites, and the amplified fragment is digested with Xba I to obtain a Hyg fragment (cut by Xba I), with a size of about 1.1 kb.

The plasmid pUCDis constructed in the previous step was digested with Xba I, and the above digested product was purified to recover a 4.0 kb fragment, which was the pUCDI fragment ( Xba I cut).

Ligate the pUCDI fragment ( Xba I cut) and Hyg fragment ( Xba I cut) to transform Escherichia coli competent DH5α, use hygromycin-resistant LB solid plate to screen positive clones, and pick single clones into LB liquid After culturing in the culture medium, the plasmid was extracted, and the correct clone identified by enzyme digestion was the plasmid pUCDH. Mutate the Kpn I site and Eco RI site in this plasmid, remove these two restriction sites, and obtain the plasmid pUCDHmKE, its sequence is shown in SEQ ID NO: 2, and its length is 426bp to 1553bp 1128bp dif- ΩHYG - dif resistance expression cassette, with HindIII restriction site aagctt and Xho I restriction site ctcgag at both ends of the resistance expression cassette. The Hyg fragment contained in the dif- ΩHYG - dif resistance expression cassette is shown in SEQ ID NO:1. The Hyg fragment removes complex structures such as transcription terminators and long natural promoters, which is convenient for PCR amplification and has fewer internal restriction sites (common restriction sites have been mutated by site-directed mutations). At the same time, Hyg comes with a short The artificial promoter can be expressed in both Escherichia coli and mycobacteria, and the transcription and translation of downstream genes will not be affected when inserted in a directional manner.

Example 2 Construction and Application of Integrated Plasmid pMH94DHmKE

The structural diagram of the integrated plasmid pMH94DHmKE is shown in Figure 4. The plasmid pMH94DHmKE contains in a clockwise direction: the LacZ promoter (which can be used for blue-white screening and to initiate the expression of the entire plasmid backbone), the phage integration site attP , and the integrase gene Int (can express integrase, so that the plasmid can be integrated into the mycobacterium genome through the attP site), dif- ΩHYG - dif resistance expression cassette, E. coli replication origin ( oriE ), ampicillin resistance gene Amp (for plasmid selection ). The two ends of the dif- ΩHYG - dif resistance expression cassette are respectively connected with the Hin dIII restriction site and the Xho I restriction site respectively, which can be used to cut out the dif- ΩHYG - dif resistance expression cassette on the plasmid.

The construction process of the integrated plasmid pMH94DHmKE is shown in Figure 5. The plasmid pMH94 used in this construction process was donated by the William Bishai Laboratory of Johns Hopkins University in the United States. The plasmid map is shown in Figure 5. For the specific construction method, see Reference 2.

The specific operation is as follows:

1. Digest the plasmid pMH94 and the plasmid pUCDHmKE constructed in Example 1 with HindIII, recover the fragment of about 6.0 kb in size digested by pMH94 and the fragment of about 1.1 kb in size obtained by digestion of pUCDHmKE, and carry out the ligation reaction of the two fragments Afterwards, transform Escherichia coli competent DH5α, use the LB solid plate containing hygromycin resistance to screen out positive clones, pick a single clone and culture it in LB liquid medium, extract the plasmid, and identify the correct clone by enzyme digestion. Plasmid pMH94DHmKE.

2. The plasmid pMH94DHmKE was transformed into Mycobacterium tuberculosis (H37Rv tuberculosis standard strain, donated by Guangzhou Chest Hospital) and Mycobacterium smegmatis (ATCC 700044) by electroporation to obtain corresponding transformants.

The resulting transformant was incubated in a 37°C incubator for 12 hours to fully revive the bacteria and express their resistance. After blowing and mixing with a gun tip with a filter element, aliquot into small tubes, centrifuge at 10,000 rpm for 1 minute to precipitate the transformed bacteria, discard the supernatant, resuspend with 0.6 mL of 7H9 medium, and spread 500 μL/plate on 7H11 containing Corresponding antibiotics (150 μg/mL HYG) culture plate. At the same time, after 100-fold dilution, 500 μL/plate was plated. Protected from light and cultured in a 37-degree incubator.

Mycobacterium tuberculosis or Mycobacterium smegmatis containing the pMH94DHmKE plasmid can be obtained on a plate containing hygromycin (HYG) . in the expression.

The obtained M. tuberculosis or M. smegmatis transformants carrying the pMH94DHmKE plasmid were subcultured in liquid, and after 3 days (for M. smegmatis) or 14 days (for M. tuberculosis) of culture, the culture The bacterial solution was diluted and plated, and about 80% of the colonies on the obtained plate lost Hyg resistance, that is, they could not grow on the plate containing hygromycin (HYG). This step proved the dif- The ΩHYG -dif resistance expression cassette can be successfully auto-dissociated in mycobacteria with high efficiency.

Example 3 Construction and application of integrated plasmid pblDHCiGn

In the integrated plasmid pblDHCiGn, the resistance expression cassette exists in the form of double restriction sites on both sides. The construction of this plasmid is to prove that the resistance expression cassette is still effective in this form. The plasmid structure is shown in Figure 6. In a clockwise direction, it contains: promoter P(BLA) (used to initiate the expression of the following Amp ), ampicillin resistance gene Amp (usable for plasmid screening), Escherichia coli origin of replication ( OriE ), phage integration site ( attP ), integrase gene Int (can express integrase, so that the plasmid can be integrated into the mycobacterium genome through the attP site), dif -ΩHYG -dif resistance expression cassette, enhanced green fluorescent protein Gene eGFP . The two ends of the dif- ΩHYG - dif resistance expression cassette were connected with different enzyme cutting sites.

The construction process of plasmid pblDHCiGn is shown in Figure 7. The plasmid pblueINT used in this process was donated by Professor Eric Nuermberger of Johns Hopkins University in the United States. Its plasmid map is shown in Figure 7. For the specific construction method, see Reference 3. NO:3 shown.

The specific construction steps are as follows:

1. Construction of plasmid pblDHC1n: Plasmid pblueINT was digested with restriction endonuclease Kpn I and Bam HI, and functional fragment A of 2.9 kb was recovered; plasmid pUCDHmKE was used as template, and primer Hygcf (5'-GCTGGTACCGCTAGCGCTGCAGCCATGGCAAGCTTCTCGAGTAAG-3') (SEQ ID NO:11) and Hygcr (5'-GCAGGATCCGATATCGAATTCCATATGCCCAAGCTTCTCGAGACT-3')(SEQ ID NO:12) amplified a 1.2 kb dif- ΩHYG - dif fragment (with Kpn I and Bam HI restriction sites at both ends, respectively) , and then digested with the functional fragment A to obtain a 4.1 kb plasmid pblDHC1n.

2. Construction of plasmid pblDHCGn: digest plasmid pblDHC1n with restriction endonucleases Nco I and Kpn I, and recover 4.1kb functional fragment B; use plasmid pEGFP-N1 (commercial plasmid) as template, use primer GYF-f (5'- GGTCCATGGTGAGCAAGGGCGAGG-3')(SEQ ID NO:13) and GYF-r(5'- GCGGGTACC TTACTTGTACAGCTCGTCCATGC-3') (SEQ ID NO:14) amplified a 734 bp eGFP fragment, which was digested with the functional fragment B is ligated to obtain plasmid pblDHCGn.

3. Construction of plasmid pblDHCiGn : digest plasmid pblDHCGn with restriction enzymes SpeI and EcoRI, and recover 4.7kb functional fragment C; digest plasmid pblueINT with restriction enzymes XbaI and EcoRI, and recover 2.1kb Int:attP fragment , and functional fragment C were connected to obtain plasmid pblDHCiGn, the sequence of which is shown in SEQ ID NO:4.

The constructed plasmid pblDHCiGn was transformed into Mycobacterium tuberculosis or Mycobacterium smegmatis by electroporation to obtain corresponding transformants, and the expression of eGFP could be observed in the transformants. This proves that after adding double restriction sites at both ends of the dif- ΩHYG - dif resistance expression cassette, the Hyg promoter can not only initiate the expression of the Hyg resistance gene, but also can activate the downstream dif sequence of the Hyg resistance gene +Expression of the restriction site and the gene behind it, where the green fluorescent egg is used as an indicator.

Next, the obtained M. tuberculosis or M. smegmatis transformants harboring the pblDHCiGn plasmid were subjected to liquid subculture after 3 days (for M. smegmatis) or 14 days (for M. tuberculosis) of culture , after diluting the cultured bacterial solution, spread it on the plate, about 80% of the colonies on the obtained plate lost HYG resistance, that is, they could not grow on the plate containing hygromycin (HYG), thus proving again that the present invention The dif- ΩHYG - dif resistance expression cassette can be successfully auto-dissociated in mycobacteria with high efficiency. Moreover, green fluorescence can still be observed in transformants that have lost HYG resistance. This design shows that the automatic dissociation of the resistance expression cassette will not affect the expression of downstream genes after its insertion, that is, the resistance designed in the present invention The presence or absence of an expression cassette does not affect the expression of downstream genes.

references:

[1] Howard NS, Gomez JE, Ko C, Bishai WR. Color selection with a hygromycin-resistance-based escherichia coli-mycobacterial shuttle vector. Gene 1995;166:181-182.

[2] Lee MH, Pascopella L, Jacobs WR, Jr., Hatfull GF. Site-specific integration of mycobacteriophage l5: Integration-proficient vectors for mycobacterium smegmatis, mycobacterium tuberculosis, and bacille S Nat U calmette-guerin. Procad 1991;88:3111-3115.

[3] Zhang T, Li SY, Nuermberger EL. Autoluminescent Mycobacterium tuberculosis for Rapid, Real-Time, Non-Invasive Assessment of Drug and Vaccine Efficacy. PLoS ONE. (2012), 7(1): e29774.

<110> Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences

<120> A resistance expression cassette for efficient construction of recombinant mycobacteria without resistance markers

<130>

<150> 2013102245916

<151> 2013-06-06

<160> 14

<170> PatentIn version 3.5

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cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 1800

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 1860

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 1920

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 1980

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 2040

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 2100

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 2160

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 2220

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 2280

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 2340

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 2400

aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 2460

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 2520

ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 2580

atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 2640

atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 2700

tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 2760

gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 2820

tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 2880

gacccacgct caccggctcc agattatca gcaataaacc agccagccgg aagggccgag 2940

cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 3000

gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 3060

atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 3120

aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 3180

atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 3240

aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 3300

aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 3360

gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 3420

gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta accactcgt 3480

gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 3540

ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 3600

ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 3660

atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 3720

gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 3780

atcacgaggc cctttcgtc 3799

the

the

<210> 3

<211> 5064

<212> DNA

<213> Artificial sequence

<400> 3

ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60

atttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120

gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180

caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240

ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300

cccccgattt agagcttgac gggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360

agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420

cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480

caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540

gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600

taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca 660

ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat tcgatatcaa 720

gcttgcatgc ctgcaggtcg accaagacga tcaccgggct cgtcggagct ggaggcgcag 780

cgggagctgg ctctgtattc tcaggcaagg ccggaggccc tggaggaaac accacggcgt 840

ccgctgtcgg atggtcaggt ttgaccgcaa ccggcggtcc cggaggctct gtgatcgaca 900

tcctcagcgt cgccggaaag tcgcctggag atcggaccta caacgaccag ctctacatag 960

gcggcgcaca acagaactca gctggcggga acggcaatgc tcctggcggc ggcggggctg 1020

gtgcccaggt ctccgcacag agcggcggtg ctggcgctcg cggccaggcg tggttcttcg 1080

cgtactgaca agaaaccccc ctctttagga ctcagtgtcc ttgggagggg ggctttttgc 1140

gtttcaggag gtcttggcca gcttggacat cgcctcagcg atagcctcgt cgcgggcctc 1200

agacgccatc tggtacttca tcgccatcct aggagtcgtg tgaccgagac gggccatcag 1260

ctccttggtc gtcgcacctg cctgagcggc gaacgtagcg ccgacagcgc ggaggtcgtg 1320

gatgcggagt tccggccgac cgatcttggc gtagccacgc ttcagcgact tggtgaacgc 1380

ggacttcgac agccggttgc cctgcgtcgt ggtcaccagg aatgcctcgg ggcccttgtt 1440

catcttcgta cggtccttca tgtgcgctcg gatcatctcc gcgacgtgag gcggaaccgt 1500

cacaggacgc ttcgaccgga cggtcttggc gttgccaacg acgatcttgt tccccacgcg 1560

ggaagcgcca cggcgcaccc ggagcttcat cgtcatgccg tcgtccacga tgtccttgcg 1620

gcgaagctcg atcagctctc cgaaccggag gctcgtccac gccaggatgt atgccgcgat 1680

ccggtagtgc tcgaagatct cagcggcgac gatgtccagc tcctcaggcg tcagcgcctc 1740

tacgtcgcgc tcatcggctg ccttctgctc gatccggcac gggttctctg cgatcagctt 1800

gtcctcgacc gctgtgttca tcaccgcccg gaggacgttg taggcatgcc ggcgggcagt 1860

cgggtgcttc ctacccatcc cggcccacca cgcacgcacc agagctggcg tcatctctgt 1920

gaccgccact tcacctagca ccgggtagat gcggcgctcc gcgtgcccgc tgtacagatc 1980

cctggtgccg tctgcgaggt cgcgctccac gagccacttc cgggtgtact cctccagcgt 2040

gatggcgctg gcggctgcct tcttcgcccg gtcctgtgga ggggtccagg tctccatctc 2100

gatgagccgc ttctcgcccg cgagccaggc ttcggcgtcc atcttgttgt cgtaggtctg 2160

cagcgcgtag tacctcacac cgtcctgcgg gttgacgtat gaggcttgga tcctcccgct 2220

gcgctgagtc ttcagcgatc cccatccgcg acgtgccaac taggtctcct ctcgtcgtga 2280

acaaggctac cgggttgcaa ctcctgtgca actctcaggc ttcaacgcgc ttctacgacc 2340

tgcaatttct ttccacttag aggatgcagc cgagagggggg taaaaaccta tcttgaccgg 2400

cccatatgtg gtcggcagac accattctt ccaaactagc tacgcgggtt cgattcccgt 2460

cgcccgctcc gctggtcaga gggtgttttc gccctctggc catttttctt tccagggggtc 2520

tgcaactctt gtgcgactct tctgacctgg gcatacgcgg ttgcaacgca tccctgatct 2580

ggctactttc gatgctgaca aacgaataga gccccccgcc tgcgcgaaca gacgaggggc 2640

attcacacca gattggagct ggtgcagtga agagaataga ccgggacaag gttgcaccgg 2700

gagttgcagc ggtcggaacc ctcgccgtcg gcgggctggc gttcgccctg tcgttcacgg 2760

ctctcagcga gctggctgcg gccaacgggg tggcccaagc agagatggtg cccttggtgg 2820

tcgactctag aggatccccg acctcgaggg ggggcccggt acccagcttt tgttcccttt 2880

agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 2940

gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 3000

gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 3060

cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 3120

tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 3180

tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 3240

ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 3300

ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 3360

gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 3420

gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 3480

ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 3540

tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 3600

gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 3660

tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 3720

tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 3780

tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3840

ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 3900

ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 3960

gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 4020

aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 4080

aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 4140

cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 4200

ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 4260

cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 4320

ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 4380

ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 4440

ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 4500

gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 4560

ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 4620

ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 4680

gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 4740

ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 4800

cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 4860

ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 4920

aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 4980

gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5040

gcacatttcc ccgaaaagtg ccac 5064

the

the

<210> 4

<211> 6898

<212> DNA

<213> Artificial sequence

<400> 4

gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60

caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120

ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180

gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240

tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300

ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360

tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420

atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480

gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540

caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600

ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660

ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720

ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggacc 780

ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840

gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900

ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960

taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020

agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080

atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140

aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200

caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260

ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320

cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380

tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440

gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500

ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560

gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620

caggagagcg cacgaggggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680

ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740

tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800

ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860

agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920

aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980

gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040

tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100

tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160

ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2220

gccgctctag aactagagtc gaccaccaag ggcaccatct ctgcttgggc caccccgttg 2280

gccgcagcca gctcgctgag agccgtgaac gacagggcga acgccagccc gccgacggcg 2340

agggttccga ccgctgcaac tcccggtgca accttgtccc ggtctattct cttcactgca 2400

ccagctccaa tctggtgtga atgcccctcg tctgttcgcg caggcggggg gctctattcg 2460

tttgtcagca tcgaaagtag ccagatcagg gatgcgttgc aaccgcgtat gcccaggtca 2520

gaagagtcgc acaagagttg cagacccctg gaaagaaaaa tggccagagg gcgaaaacac 2580

cctctgacca gcggagcggg cgacgggaat cgaacccgcg tagctagttt ggaagaatgg 2640

gtgtctgccg accacatatg ggccggtcaa gataggtttt taccccctct cggctgcatc 2700

ctctaagtgg aaagaaattg caggtcgtag aagcgcgttg aagcctgaga gttgcacagg 2760

agttgcaacc cggtagcctt gttcacgacg agaggagacc tagttggcac gtcgcggatg 2820

gggatcgctg aagactcagc gcagcgggag gatccaagcc tcatacgtca acccgcagga 2880

cggtgtgagg tactacgcgc tgcagaccta cgacaacaag atggacgccg aagcctggct 2940

cgcgggcgag aagcggctca tcgagatgga gacctggacc cctccacagg accgggcgaa 3000

gaaggcagcc gccagcgcca tcacgctgga ggagtacacc cggaagtggc tcgtggagcg 3060

cgacctcgca gacggcacca gggatctgta cagcgggcac gcggagcgcc gcatctaccc 3120

ggtgctaggt gaagtggcgg tcacagagat gacgccagct ctggtgcgtg cgtggtgggc 3180

cgggatgggt aggaagcacc cgactgcccg ccggcatgcc tacaacgtcc tccgggcggt 3240

gatgaacaca gcggtcgagg acaagctgat cgcagagaac ccgtgccgga tcgagcagaa 3300

ggcagccgat gagcgcgacg tagaggcgct gacgcctgag gagctggaca tcgtcgccgc 3360

tgagatcttc gagcactacc ggatcgcggc atacatcctg gcgtggacga gcctccggtt 3420

cggagagctg atcgagcttc gccgcaagga catcgtggac gacggcatga cgatgaagct 3480

ccgggtgcgc cgtggcgctt cccgcgtggg gaacaagatc gtcgttggca acgccaagac 3540

cgtccggtcg aagcgtcctg tgacggttcc gcctcacgtc gcggagatga tccgagcgca 3600

catgaaggac cgtacgaaga tgaacaaggg ccccgaggca ttcctggtga ccacgacgca 3660

gggcaaccgg ctgtcgaagt ccgcgttcac caagtcgctg aagcgtggct acgccaagat 3720

cggtcggccg gaactccgca tccacgacct ccgcgctgtc ggcgctacgt tcgccgctca 3780

ggcaggtgcg acgaccaagg agctgatggc ccgtctcggt cacacgactc ctaggatggc 3840

gatgaagtac cagatggcgt ctgaggcccg cgacgaggct atcgctgagg cgatgtccaa 3900

gctggccaag acctcctgaa acgcaaaaag cccccctccc aaggacactg agtcctaaag 3960

aggggggttt cttgtcagta cgcgaagaac cacgcctggc cgcgagcgcc agcaccgccg 4020

ctctgtgcgg agacctgggc accagccccg ccgccgccag gagcattgcc gttcccgcca 4080

gctgagttct gttgtgcgcc gcctatgtag agctggtcgt tgtaggtccg atctccaggc 4140

gactttccgg cgacgctgag gatgtcgatc acaggcctc cgggaccgcc ggttgcggtc 4200

aaacctgacc atccgacagc ggacgccgtg gtgtttcctc cagggcctcc ggccttgcct 4260

gagaatacag agccagctcc cgctgcgcct ccagctccga cgagcccggt gatcgtcttg 4320

gtcgacctgc aggcatgcaa gcttgatatc gaattccata tgcccaagct tctcgagact 4380

tgacataatg tcgcttatcg gcttaatcga tctagaccgg ccgtgcggaa ttaagccggc 4440

ccgtaccctg tgaatagagg tccgctgtga cacaagaatc cctgttactt ctcgaccgta 4500

ttgattcgga tgattcctac gcgagcctgc ggaacgacca ggagttctgg gagccgctgg 4560

cccgccgagc cctggaggag ctcgggctgc cggtgccgcc ggtgctgcgg gtgcccggcg 4620

agagcaccaa ccccgtactg gtcggcgagc ccggcccggt gatcaagctg ttcggcgagc 4680

actggtgcgg tccggagagc ctcgcgtcgg agtcggaggc gtacgcggtc ctggcggacg 4740

ccccggttcc ggtgccccgc ctcctcggcc gcggcgagct gcggcccggc accggagcct 4800

ggccgtggcc ctacctggtg atgagccgga tgaccggcac cacctggcgg tccgcgatgg 4860

acggcacgac cgaccggaac gcgctgctcg ccctggcccg cgaactcggc cgggtgctcg 4920

gacggctgca cagggtgccg ctgaccggga acaccgtgct cacccccccat tccgaggtct 4980

tcccggaact gctgcgggaa cgccgcgcgg cgaccgtcga ggaccaccgc gggtggggct 5040

acctctcgcc ccggctgctg gaccgcctgg aggactggct gccggacgtg gacacgctgc 5100

tggccggccg cgaaccccgg ttcgtccacg gcgacctgca cgggaccaac atcttcgtgg 5160

acctggccgc gaccgaggtc accgggatcg tcgacttcac cgacgtctat gcgggagact 5220

cccgctacag cctggtgcaa ctgcatctca acgccttccg gggcgaccgc gagatcctgg 5280

ccgcgctgct cgacggggcg cagtggaagc ggaccgagga cttcgcccgc gaactgctcg 5340

ccttcacctt cctgcacgac ttcgaggtgt tcgaggagac cccgctggat ctctccggct 5400

tcaccgatcc ggaggaactg gcgcagttcc tctgggggcc gccggacacc gcccccggcg 5460

cctgatctag acccgggact tgacataatg tcgcttatcg gcttactcga gaagcttgcc 5520

atggtgagca agggcgagga gctgttcacc gggtggtgc ccatcctggt cgagctggac 5580

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 5640

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 5700

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 5760

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 5820

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 5880

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 5940

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 6000

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 6060

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 6120

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 6180

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 6240

ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca 6300

acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 6360

tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 6420

cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 6480

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 6540

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 6600

ccctttaggg ttccgatta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 6660

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 6720

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 6780

ggtctattct tttgattatt aagggatttt gccgatttcg gcctattggt taaaaaatga 6840

gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttag 6898

the

the

<210> 5

<211> 1152

<212> DNA

<213> Artificial sequence

<400> 5

aagcttctcg agacttgaca taatgtcgct tatcggctta atcgatctag accggccgtg 60

cggaattaag ccggcccgta ccctgtgaat agaggtccgc tgtgacacaa gaatccctgt 120

tacttctcga ccgtattgat tcggatgatt cctacgcgag cctgcggaac gaccaggagt 180

tctgggagcc gctggcccgc cgagccctgg aggagctcgg gctgccggtg ccgccggtgc 240

tgcgggtgcc cggcgagagc accaaccccg tactggtcgg cgagcccggc ccggtgatca 300

agctgttcgg cgagcactgg tgcggtccgg agagcctcgc gtcggagtcg gaggcgtacg 360

cggtcctggc ggacgccccg gttccggtgc cccgcctcct cggccgcggc gagctgcggc 420

ccggcaccgg agcctggccg tggccctacc tggtgatgag ccggatgacc ggcaccacct 480

ggcggtccgc gatggacggc acgaccgacc ggaacgcgct gctcgccctg gcccgcgaac 540

tcggccgggt gctcggacgg ctgcacaggg tgccgctgac cgggaacacc gtgctcaccc 600

cccattccga ggtcttcccg gaactgctgc gggaacgccg cgcggcgacc gtcgaggacc 660

accgcgggtg gggctacctc tcgccccggc tgctggaccg cctggaggac tggctgccgg 720

acgtggacac gctgctggcc ggccgcgaac cccggttcgt ccacggcgac ctgcacggga 780

ccaacatctt cgtggacctg gccgcgaccg aggtcaccgg gatcgtcgac ttcaccgacg 840

tctatgcggg agactcccgc tacagcctgg tgcaactgca tctcaacgcc ttccggggcg 900

accgcgagat cctggccgcg ctgctcgacg gggcgcagtg gaagcggacc gaggacttcg 960

cccgcgaact gctcgccttc accttcctgc acgacttcga ggtgttcgag gagaccccgc 1020

tggatctctc cggcttcacc gatccggagg aactggcgca gttcctctgg gggccgccgg 1080

acaccgcccc cggcgcctga tctagacccg ggacttgaca taatgtcgct tatcggctta 1140

ctcgagaagc tt 1152

the

the

<210> 6

<211> 1226

<212> DNA

<213> Artificial sequence

<400> 6

gcggccgctc tagaactagt ggatccgata tcgaattcca tatgcccaag cttctcgaga 60

cttgacataa tgtcgcttat cggcttaatc gatctagacc ggccgtgcgg aattaagccg 120

gcccgtaccc tgtgaataga ggtccgctgt gacacaagaa tccctgttac ttctcgaccg 180

tattgattcg gatgattcct acgcgagcct gcggaacgac caggagttct gggagccgct 240

ggcccgccga gccctggagg agctcgggct gccggtgccg ccggtgctgc gggtgcccgg 300

cgagagcacc aaccccgtac tggtcggcga gcccggcccg gtgatcaagc tgttcggcga 360

gcactggtgc ggtccggaga gcctcgcgtc ggagtcggag gcgtacgcgg tcctggcgga 420

cgccccggtt ccggtgcccc gcctcctcgg ccgcggcgag ctgcggcccg gcaccggagc 480

ctggccgtgg ccctacctgg tgatgagccg gatgaccggc accacctggc ggtccgcgat 540

ggacggcacg accgaccgga acgcgctgct cgccctggcc cgcgaactcg gccgggtgct 600

cggacggctg cacagggtgc cgctgaccgg gaacaccgtg ctcacccccc attccgaggt 660

cttcccggaa ctgctgcggg aacgccgcgc ggcgaccgtc gaggaccacc gcgggtgggg 720

ctacctctcg ccccggctgc tggaccgcct ggaggactgg ctgccggacg tggacacgct 780

gctggccggc cgcgaaccccc ggttcgtcca cggcgacctg cacgggacca acatcttcgt 840

ggacctggcc gcgaccgagg tcaccgggat cgtcgacttc accgacgtct atgcgggaga 900

ctcccgctac agcctggtgc aactgcatct caacgccttc cggggcgacc gcgagatcct 960

ggccgcgctg ctcgacgggg cgcagtggaa gcggaccgag gacttcgccc gcgaactgct 1020

cgccttcacc ttcctgcacg acttcgaggt gttcgaggag accccgctgg atctctccgg 1080

cttcaccgat ccggaggaac tggcgcagtt cctctggggg ccgccggaca ccgcccccgg 1140

cgcctgatct agacccggga cttgacataa tgtcgcttat cggcttactc gagaagcttg 1200

gccatggctg cagcgctagc ggtacc 1226

the

the

<210> 7

<211> 51

<212> DNA

<213> Artificial sequence

<400> 7

agcttctcga gtaagccgat aagcgacatt atgtcaagtc ccgggtctag a 51

the

the

<210> 8

<211> 51

<212> DNA

<213> Artificial sequence

<400> 8

atcgattaag ccgataagcg aattatgtc aagtctcgag aagcttggta c 51

the

the

<210> 9

<211> 26

<212> DNA

<213> Artificial sequence

<400> 9

tgtctagacc ggccgtgcgg aattaa 26

the

the

<210> 10

<211> 29

<212> DNA

<213> Artificial sequence

<400> 10

attctagatc aggcgccggg ggcggtgtc 29

the

the

<210> 11

<211> 45

<212> DNA

<213> Artificial sequence

<400> 11

gctggtaccg ctagcgctgc agccatggca agcttctcga gtaag 45

the

the

<210> 12

<211> 45

<212> DNA

<213> Artificial sequence

<400> 12

gcaggatccg atatcgaatt ccatatgccc aagcttctcg agact 45

the

the

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence

<400> 13

ggtccatggt gagcaagggc gagg 24

the

the

<210> 14

<211> 32

<212> DNA

<213> Artificial sequence

<400> 14

gcgggtacct tacttgtaca gctcgtccat gc 32

Claims (10)

1. simplify an improved hygromycin gene, its nucleotide sequence is as shown in SEQ ID NO:1.

2. for efficiently building resistance expression's box of non-resistant mark recombinant mycobacterium, containing the hygromycin gene after simplifying, and be positioned at hygromycin gene two ends dif1with dif2sequence, is characterized in that, described in simplify after the nucleotide sequence of hygromycin gene as shown in SEQ ID NO:1, dif1with dif2sequence is respectively as shown in SEQ ID NO:7 and SEQ ID NO:8.

3. resistance expression's box according to claim 2, is characterized in that, described expression cassette two sections also added multiple restriction enzyme site.

4. resistance expression's box according to claim 3, is characterized in that, the nucleotide sequence of described resistance expression's box is as shown in SEQ ID NO:5 or SEQ ID NO:6.

5. the recombinant plasmid containing resistance expression's box described in any one of claim 2 ~ 4.

6. recombinant plasmid according to claim 5, is characterized in that, the nucleotide sequence of described plasmid is as shown in SEQ ID NO:2.

7., for efficiently building a recombinant plasmid for non-resistant mark recombinant mycobacterium, it contains: promotor, phage integration site, integrase gene, the resistance expression's box described in any one of claim 2 ~ 4, replication origin.

8. recombinant plasmid according to claim 7, is characterized in that, in the direction of the clock, successively containing phage integration site, integrase gene, resistance expression's box described in any one of claim 2 ~ 4.

9. the recombinant plasmid according to claim 7 or 8, is characterized in that, described promotor is LacZ promotor or BLA promotor, and described replication origin is intestinal bacteria replication orgin.

10. the recombinant plasmid according to claim 7 or 8, is characterized in that, the nucleotide sequence of described recombinant plasmid is as shown in SEQ ID NO:4.