Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V008167 | pCRISPR-Cas9 | 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:
- pCRISPR-Cas9
- Antibiotic Resistance:
- Apramycin
- Length:
- 11265 bp
- Type:
- Actinomyces vector
- Replication origin:
- ori
- Source/Author:
- Tong Y, Charusanti P, Zhang L, Weber T, Lee SY.
pCRISPR-Cas9 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.
pCRISPR-Cas9 vector Sequence
LOCUS V008167 11265 bp DNA circular SYN 17-DEC-2018
DEFINITION Exported.
ACCESSION V008167
VERSION V008167
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 11265)
AUTHORS Tong Y, Charusanti P, Zhang L, Weber T, Lee SY.
TITLE CRISPR-Cas9 based engineering of actinomycetal genomes
JOURNAL ACS Synth Biol (2015) In press
PUBMED 25806970
REFERENCE 2 (bases 1 to 11265)
AUTHORS Tong Y, Charusanti P, Zhang L, Weber T, Lee SY.
TITLE Direct Submission
JOURNAL Submitted (23-MAR-2015) The Novo Nordisk Foundation Center for
Biosustainability, Kogle Alle 6, Horsholm 2970, Denmark
REFERENCE 3 (bases 1 to 11265)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 11265)
AUTHORS .
TITLE Direct Submission
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth
Biol (2015) In press"
SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(23-MAR-2015) The Novo Nordisk Foundation Center for
Biosustainability, Kogle Alle 6, Horsholm 2970, Denmark"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..11265
/mol_type="other DNA"
/organism="synthetic DNA construct"
oriT complement(467..576)
/direction=LEFT
/label="oriT"
/note="incP origin of transfer"
CDS 892..1692
/label="ApmR"
/note="aminoglycoside 3-N-acetyltransferase type IV"
rep_origin 1994..2582
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 2870..2891
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2906..2936
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 2944..2960
/label="lac operator"
/bound_moiety="lac repressor encoded by lacI"
/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)."
primer_bind 2968..2984
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
misc_RNA 3123..3198
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator complement(3368..3402)
/label="lambda t0 terminator"
/note="minimal transcription terminator from phage lambda
(Scholtissek and Grosse, 1987)"
CDS 3570..7673
/label="Cas9"
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
terminator complement(7863..7911)
/label="fd terminator"
/note="central terminator from bacteriophage fd (Otsuka and
Kunisawa, 1982)"
CDS 8255..9061
/gene="tsnR"
/label="23S rRNA (adenosine(1067)-2'-O)-methyltransferase"
/note="23S rRNA (adenosine(1067)-2'-O)-methyltransferase
from Streptomyces azureus. Accession#: P18644"
CDS 9627..11066
/label="pSG5 Rep"
/note="replication initiator protein from the Streptomyces
ghanaensis plasmid pSG5"