Price Information
Cat No. | Plasmid Name | Availability | Add to cart |
---|---|---|---|
V006723 | pET-28b-Cas9-His | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pET-28b-Cas9-His
- Antibiotic Resistance:
- Kanamycin
- Length:
- 9375 bp
- Type:
- Bacterial Expression, CRISPR
- Replication origin:
- ori
- Copy Number:
- High Copy
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- T7
pET-28b-Cas9-His 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.
pET-28b-Cas9-His vector Sequence
LOCUS pET-28b-Cas9-His 9375 bp DNA circular SYN 13-MAY-2021 DEFINITION For in vitro expression and purification of Cas9 protein. ACCESSION . VERSION . KEYWORDS pET-28b-Cas9-His SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9375) AUTHORS Gagnon JA, Valen E, Thyme SB, Huang P, Ahkmetova L, Pauli A, Montague TG, Zimmerman S, Richter C, Schier AF TITLE Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. JOURNAL PLoS One. 2014 May 29;9(5):e98186. doi: 10.1371/journal.pone.0098186. eCollection 2014. PUBMED 24873830 REFERENCE 2 (bases 1 to 9375) TITLE Direct Submission REFERENCE 3 (bases 1 to 9375) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One."; date: "2014-05-29"; pages: " 10.1371/journal.pone.0098186. eCollection 2014" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..9375 /mol_type="other DNA" /organism="synthetic DNA construct" misc_feature complement(5..144) /label=bom /note="basis of mobility region from pBR322" primer_bind 230..252 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(249..437) /label=rop /note="Rop protein, which maintains plasmids at low copy number" protein_bind complement(1212..1233) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS complement(1249..2328) /label=lacI /note="lac repressor" promoter complement(2329..2406) /label=lacI promoter primer_bind 2612..2631 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" promoter 2715..2733 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 2734..2758 /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)." RBS 2773..2795 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 2802..6905 /label=Cas9 /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" CDS 6918..6938 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6954..6971 /label=6xHis /note="6xHis affinity tag" terminator 7038..7085 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 7122..7577 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" CDS complement(7672..8484) /label=KanR /note="aminoglycoside phosphotransferase" rep_origin 8606..9194 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9348..9365 /label=L4440 /note="L4440 vector, forward primer"