pNIT-1 vector (V004308)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V004308	pNIT-1	In stock, instant shipping	Buy one, get one free!

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

Basic Vector Information

Vector Name:: pNIT-1

Antibiotic Resistance:: Kanamycin

Length:: 6207 bp

Type:: Expression vector

Replication origin:: ori

Source/Author:: Pandey AK, Raman S, Proff R, Joshi S, Kang CM, Rubin EJ, Husson RN, Sassetti CM.

pNIT-1 vector Map

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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.

pNIT-1 vector Sequence

LOCUS       40924_33292        6207 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIT-1, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6207)
  AUTHORS   Pandey AK, Raman S, Proff R, Joshi S, Kang CM, Rubin EJ, Husson RN, 
            Sassetti CM.
  TITLE     Nitrile-inducible gene expression in mycobacteria
  JOURNAL   Tuberculosis (Edinb) 89 (1), 12-16 (2009)
  PUBMED    18801704
REFERENCE   2  (bases 1 to 6207)
  AUTHORS   Pandey AK, Proff R, Joshi S, Sassetti CM.
  TITLE     Direct Submission
  JOURNAL   Submitted (01-SEP-2008) Molecular Genetics and Microbiology, 
            University of Massachusetts Medical School, 55 Lake Avenue North. 
            S6-141, Worcester, MA 01655, USA
REFERENCE   3  (bases 1 to 6207)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6207)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Tuberculosis (Edinb)"; date: "2009"; volume: "89"; issue: "1"; 
            pages: "12-16"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (01-SEP-2008) Molecular Genetics and Microbiology, University of 
            Massachusetts Medical School, 55 Lake Avenue North. S6-141, 
            Worcester, MA 01655, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             141..953
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      1288..1876
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      2035..3930
                     /label=pAL5000 mycobacterial origin of replication
                     /note="pAL5000 mycobacterial origin of replication"
     terminator      complement(3974..4021)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     regulatory      4059..4173
                     /label=PnitA
                     /note="PnitA"
                     /regulatory_class="promoter"
     misc_feature    4174..4222
                     /label=multiple cloning site
                     /note="multiple cloning site"
     terminator      4229..4268
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     regulatory      4276..4390
                     /label=PnitA
                     /note="PnitA"
                     /regulatory_class="promoter"
     CDS             4391..5350
                     /codon_start=1
                     /gene="nitR"
                     /product="nitrile-inducible regulatory protein"
                     /label=nitR
                     /protein_id="ACI22356.1"
                     /translation="MNTFFSSDQVSAPDRVALWHDVICRSYVPLNITLTSEQPFIGTVS
                     TGNLGTVRIATSSSLPQQITRTRRLIRQDEREYLMVGVQSAGHALVQQHGRTARVGRGG
                     LVFWDTRHPYDILFPTDWRMSVFQFPRYSFGFTEDFIGRMTAVNVGGDRGIGRVVSSFM
                     TSINDATDAGDLAEVASLHNSAVDLLSAAIRTELADQAAASDGLLECVLAYIRQNLADP
                     NLCASQIAAEHNVSVRTLHRLFSATGQGVAEHIRNLRLERIKTELADPTSRRYTISALA
                     RKWGFLDPSTFSRAFKDAYGITAREWAASASASPTEVS"
     gene            4391..5350
                     /gene="nitR"
                     /label=nitR
     terminator      complement(5388..5415)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(5507..5593)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"