Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V012764 | pwtCas9-bacteria | In stock, 1 week for quality controls |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
aTc-inducible expression of wild-type Cas9 (S .pyogenes) for bacterial gene knockdown
- Vector Name:
- pwtCas9-bacteria
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6932 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Stanley Qi
- Copy Number:
- High copy number
- Promoter:
- tetR/tetAs
- Expression Method:
- aTc-inducible
pwtCas9-bacteria 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.
pwtCas9-bacteria vector Sequence
LOCUS pwtCas9-bacteria 6932 bp DNA circular SYN 18-SEP-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS pwtCas9-bacteria
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6932)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 6932)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6932
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(10..633)
/label=TetR
/note="tetracycline repressor TetR"
promoter 649..704
/label=tetR/tetA promoters
/note="overlapping promoters for bacterial tetR and tetA"
RBS 722..733
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
CDS 740..4843
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
terminator 4870..4941
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 4957..4984
/label=T7Te terminator
/note="phage T7 early transcription terminator"
rep_origin complement(5151..5739)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
terminator complement(5827..5921)
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
CDS complement(5954..6811)
/label=AmpR
/note="beta-lactamase"
promoter complement(6812..6916)
/label=AmpR promoter