Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V015086 | pCasKP-Ery | 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:
- pCasKP-Ery
- Antibiotic Resistance:
- Erythromycin
- Length:
- 11656 bp
- Type:
- Genome editing
- Replication origin:
- pSC101 ori
- Host:
- E. coli
- Copy Number:
- Low
- Promoter:
- araBAD
- 5' Primer:
- G27066-F1
- Growth Temperature:
- 30℃
pCasKP-Ery vector 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.
pCasKP-Ery vector Sequence
LOCUS V015086 11656 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V015086
VERSION V015086
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 11656)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..11656
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..285
/label="araBAD promoter"
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
CDS 344..757
/label="Gam"
/note="inhibitor of the host RecBCD nuclease in the lambda
Red system"
CDS 766..1548
/label="Beta"
/note="single-stranded DNA binding recombinase in the
lambda Red system"
CDS 1548..2225
/label="Exo"
/note="5' to 3' double-stranded DNA exonuclease in the
lambda Red system"
CDS 2810..6913
/label="Cas9"
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS complement(7054..8001)
/label="Rep101(Ts)"
/note="temperature-sensitive version of the RepA protein
needed for replication with the pSC101 origin (Armstrong et
al., 1984)"
rep_origin complement(8049..8271)
/direction=LEFT
/label="pSC101 ori"
/note="low-copy replication origin that requires the Rep101
protein"
promoter 8901..9003
/label="cat promoter"
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS 9785..10519
/gene="ermBP"
/label="rRNA adenine N-6-methyltransferase"
/note="rRNA adenine N-6-methyltransferase from Enterococcus
faecalis. Accession#: P0A4D5"
CDS complement(10755..11630)
/label="araC"
/note="L-arabinose regulatory protein"