Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V016695 | pKCcas9do | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pKCcas9do
- Antibiotic Resistance:
- Apramycin
- Length:
- 13375 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- Streptomyces
- Promoter:
- J23119(SpeI)
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 30℃
pKCcas9do 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.
pKCcas9do vector Sequence
LOCUS V016695 13375 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V016695
VERSION V016695
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 13375)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..13375
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(550..1989)
/label="pSG5 Rep"
/note="replication initiator protein from the Streptomyces
ghanaensis plasmid pSG5"
oriT 3156..3265
/label="oriT"
/note="incP origin of transfer"
CDS 3298..3666
/label="traJ"
/note="oriT-recognizing protein"
CDS 4464..5207
/gene="actIII"
/label="Putative ketoacyl reductase"
/note="Putative ketoacyl reductase from Streptomyces
coelicolor (strain ATCC BAA-471 / A3(2) / M145).
Accession#: P16544"
misc_RNA complement(6428..6503)
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(6524..6558)
/label="J23119(SpeI) promoter"
/note="bacterial promoter (Registry of Standard Biological
Parts BBa_J23119) modified to end with an SpeI site"
CDS 6770..10873
/label="Cas9"
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
promoter complement(10939..10969)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(10984..11005)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(11293..11881)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 12085..12885
/label="ApmR"
/note="aminoglycoside 3-N-acetyltransferase type IV"