Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V016659 | pNICKclos1.0 | 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:
- pNICKclos1.0
- Antibiotic Resistance:
- Ampicillin
- Length:
- 11082 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- Clostridium
- Selection Marker:
- Ery
- Promoter:
- J23119(SpeI)
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
pNICKclos1.0 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.
pNICKclos1.0 vector Sequence
LOCUS V016659 11082 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V016659
VERSION V016659
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 11082)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..11082
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 14..48
/label="J23119(SpeI) promoter"
/note="bacterial promoter (Registry of Standard Biological
Parts BBa_J23119) modified to end with an SpeI site"
misc_RNA 69..144
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS 2304..6407
/label="Cas9(D10A)"
/note="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
promoter 6912..7016
/label="AmpR promoter"
CDS 7017..7874
/label="AmpR"
/note="beta-lactamase"
rep_origin 8048..8636
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 8924..8945
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 8960..8990
/label="lac promoter"
/note="promoter for the E. coli lac operon"
CDS 9406..10137
/gene="ermC'"
/label="rRNA adenine N-6-methyltransferase"
/note="rRNA adenine N-6-methyltransferase from Bacillus
subtilis. Accession#: P13956"