Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007304 | pKMD106e - intein-Cas9(S219) | 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:
- pKMD106e - intein-Cas9(S219)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8850 bp
- Type:
- Mammalian Expression, CRISPR
- Replication origin:
- ori
- Promoter:
- CMV
- 5' Primer:
- CMV-F
- 3' Primer:
- OK569
pKMD106e - intein-Cas9(S219) 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.
pKMD106e - intein-Cas9(S219) vector Sequence
LOCUS V007304 8850 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V007304
VERSION V007304
KEYWORDS pKMD106e - intein-Cas9(S219)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8850)
AUTHORS Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR
TITLE Small molecule-triggered Cas9 protein with improved genome-editing
specificity.
JOURNAL Nat Chem Biol. 2015 May;11(5):316-8. doi: 10.1038/nchembio.1793.
Epub 2015 Apr 6.
PUBMED 25848930
REFERENCE 2 (bases 1 to 8850)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8850)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/nchembio.1793"; journalName: "Nat Chem Biol"; date:
"2015-05"; volume: "11"; issue: "5"; pages: "316-8"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8850
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 132..335
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 377..395
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 403..412
/label="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 1660..2022
/gene="ESR1"
/label="Estrogen receptor"
/note="Estrogen receptor from Macaca mulatta. Accession#:
P49886"
CDS 3250..5748
/label="Cas9(C)"
/note="C-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
CDS 5755..5775
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 5782..5847
/label="3xFLAG"
/note="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
CDS 5865..5882
/label="6xHis"
/note="6xHis affinity tag"
polyA_signal 5911..6135
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(6206..6222)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6230..6246)
/label="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(6254..6284)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(6299..6320)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6437..6454)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(6608..7196)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7370..8227)
/label="AmpR"
/note="beta-lactamase"
promoter complement(8228..8332)
/label="AmpR promoter"
primer_bind complement(8411..8430)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 8602..8850
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"