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pCRISPR vector (V006962)

Price Information

Cat No. Plasmid Name Availability Add to cart
V006962 pCRISPR In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
pCRISPR
Antibiotic Resistance:
Kanamycin
Length:
2707 bp
Type:
Bacterial Expression, CRISPR ; E.coli
Replication origin:
ori
Copy Number:
High Copy
Promoter:
PLtetO-1

pCRISPR vector Map

Copy Sequence View Map
pCRISPR2707 bp600120018002400pBRforEcoNeoR/KanRlambda t0 terminatororirrnB T1 terminatorcrRNA leaderDRDRcrRNA leaderDRDRPLtetO-1 promoter

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.

pCRISPR vector Sequence

LOCUS       40924_13440        2707 bp DNA     circular SYN 13-MAY-2021
DEFINITION  A crRNA expression plasmid for targeting a specific sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2707)
  AUTHORS   Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA
  TITLE     RNA-guided editing of bacterial genomes using CRISPR-Cas systems.
  JOURNAL   Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2508.
  PUBMED    23360965
REFERENCE   2  (bases 1 to 2707)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 2707)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2508."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(15..33)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     CDS             212..1003
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     terminator      1037..1131
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1219..1807
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(1972..2058)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     misc_feature    2088..2219
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   2220..2255
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   2286..2321
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes
                     CRISPR/Cas system"
     misc_feature    2362..2493
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   2494..2529
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   2560..2595
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes
                     CRISPR/Cas system"
     promoter        complement(2628..2701)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator
                     sites (Lutz and Bujard, 1997)"