Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012338 | HBT-pcoCas9 | 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:
- HBT-pcoCas9
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7940 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM,
- Copy Number:
- High copy number
- Promoter:
- 35SPPDK
HBT-pcoCas9 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.
HBT-pcoCas9 vector Sequence
LOCUS HBT-pcoCas9. 7940 bp DNA circular SYN 01-JAN-1980
DEFINITION Plasmid for transient expression of plant codon-optimized Cas9 in
plant cells. Use together with pUC119-gRNA.
ACCESSION .
VERSION .
KEYWORDS HBT-pcoCas9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7940)
AUTHORS Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM,
Sheen J.
TITLE Multiplex and homologous recombination-mediated genome editing in
Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.
JOURNAL Nat. Biotechnol. 2013;31:688-91.
PUBMED 23929339
REFERENCE 2 (bases 1 to 7940)
AUTHORS Sheen Lab / Addgene #52254
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7940)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat.
Biotechnol."; date: "2013"; volume: "31"; pages: "688-91"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7940
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(39..55)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(63..79)
/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(87..117)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(132..153)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(441..1029)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(1203..2060)
/label=AmpR
/note="beta-lactamase"
promoter complement(2061..2165)
/label=AmpR promoter
primer_bind 2639..2655
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 2722..3231
/label=35SPPDK promoter
/note="hybrid promoter consisting of the cauliflower mosaic
virus 35S enhancer fused to the maize C4PPDK basal promoter
(Yoo et al., 2007)"
CDS 3240..3242
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 3243..3290
/label=2xFLAG
/note="two tandem FLAG(R) epitope tags"
CDS 3297..3317
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
intron 4779..4967
/label=IV2 intron
/note="modified second intron of the potato ST-LS1 gene
(Vancanneyt et al., 1990)"
CDS 5133..7631
/label=Cas9(C)
/note="C-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
CDS 7632..7679
/label=nucleoplasmin NLS
/note="bipartite nuclear localization signal from
nucleoplasmin"
terminator 7688..7940
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"