pMV261 vector (V012795)

Price Information

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Plasmid DNA pMV261 is a replicative vector (multicopy, extrachromosomal) that contains a DNA cassette encoding kanamycin resistance (Tn903-derived aph gene), an E. coli origin of replication (oriE), a mycobacterial plasmid DNA origin of replication (oriM), an expression cassette containing a mycobacterial promoter, a multiple cloning site, and a transcriptional terminator.

pMV261 vector Map

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

• Ding GQ, Yu YL, Shen ZJ, Zhou XL, Chen SW, Liao GD, Zhang Y. Antitumor effects of human interferon-alpha 2b secreted by recombinant bacillus Calmette-Guérin vaccine on bladder cancer cells. J Zhejiang Univ Sci B. 2012 May;13(5):335-41. doi: 10.1631/jzus.B1100366. PMID: 22556170; PMCID: PMC3348223.
• Xue QJ, Li YQ, Yang CQ, Chen T, Li XZ, Cheng B, Wang CM. Anti-tumour research of recombinant BCG using BZLF1 and hGM-CSF fusion genes. Vaccine. 2017 Mar 14;35(12):1599-1607. doi: 10.1016/j.vaccine.2017.02.024. Epub 2017 Feb 20. PMID: 28228322.
• Gu L, Chen Y, Wang Q, Li X, Mi K, Deng H. Functional Characterization of Sirtuin-like Protein in Mycobacterium smegmatis. J Proteome Res. 2015 Nov 6;14(11):4441-9. doi: 10.1021/acs.jproteome.5b00359. Epub 2015 Sep 29. PMID: 26375486.

pMV261 vector Sequence

LOCUS       Exported                4494 bp DNA     circular SYN 20-NOV-2024
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pMV261
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4494)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4494)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4494)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4494)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4494
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          2035..2048
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             139..951
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      1285..1873
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      2032..3933
                     /label=Mycobacterial OriM region derived from pAL5000
                     /note="Mycobacterial OriM region derived from pAL5000"
     promoter        3965..4353
                     /label=Hsp60 promoter
                     /note="HSP60 promoter from Mycobacterium tuberculosis."
     terminator      4426..4472
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"