Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012806 | pKEW303-AcrVA5 | 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
Expresses AcrVA5 in bacteria, under TetR control.
- Vector Name:
- pKEW303-AcrVA5
- Antibiotic Resistance:
- Ampicillin
- Length:
- 3198 bp
- Type:
- E.coli expression plasmid
- Replication origin:
- ori
- Source/Author:
- Jennifer Doudna
- Copy Number:
- High copy number
- Promoter:
- tetR/tetAs
pKEW303-AcrVA5 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.
pKEW303-AcrVA5 vector Sequence
LOCUS pKEW303-AcrVA5. 3198 bp DNA circular SYN 31-AUG-2022
DEFINITION Expresses AcrVA5 in bacteria, under TetR control..
ACCESSION .
VERSION .
KEYWORDS pKEW303-AcrVA5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3198)
AUTHORS Watters KE, Fellmann C, Bai HB, Ren SM, Doudna JA
TITLE Systematic discovery of natural CRISPR-Cas12a inhibitors.
JOURNAL Science. 2018 Oct 12;362(6411):236-239. doi:
10.1126/science.aau5138. Epub 2018 Sep 6.
PUBMED 30190307
REFERENCE 2 (bases 1 to 3198)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1126/science.aau5138"; journalName: "Science"; date:
"2018-10-12- 12"; volume: "362"; issue: "6411"; pages: "236-239"
FEATURES Location/Qualifiers
source 1..3198
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..56
/label=tetR/tetA promoters
/note="overlapping promoters for bacterial tetR and tetA"
CDS 83..361
/codon_start=1
/label=anti-CRISPR N-acetyltransferase AcrVA5
/note="anti-CRISPR N-acetyltransferase AcrVA5"
/translation="MKIELSGGYICYSIEEDEVTIDMVEVTTKRQGIGSQLIDMVKDVA
REVGLPIGLYAYPQDDSISQEDLIEFYFSNDFEYDPDDVDGRLMRWS"
terminator 594..621
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
rep_origin complement(747..1335)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(1509..2366)
/label=AmpR
/note="beta-lactamase"
promoter complement(2367..2471)
/label=AmpR promoter
CDS complement(2560..3183)
/label=TetR
/note="tetracycline repressor TetR"