Basic Vector Information
- Vector Name:
- pwtcas9
- Antibiotic Resistance:
- Streptomycin
- Length:
- 7692 bp
- Type:
- Cloning vector
- Replication origin:
- CloDF13 ori
- Source/Author:
- Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J, Turczyk BM, Tung M, Collins JJ, Church GM.
- Promoter:
- tetR/tetAs
pwtcas9 vector Vector Map
Plasmid Resuspension 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 ℃.
pwtcas9 vector Sequence
LOCUS 40924_46658 7692 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pwtcas9, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7692)
AUTHORS Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J,
Turczyk BM, Tung M, Collins JJ, Church GM.
TITLE Precise Cas9 targeting enables genomic mutation prevention
JOURNAL Proc. Natl. Acad. Sci. U.S.A. (2018) In press
PUBMED 29555762
REFERENCE 2 (bases 1 to 7692)
AUTHORS Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J,
Turczyk BM, Tung M, Collins JJ, Church GM.
TITLE Direct Submission
JOURNAL Submitted (02-MAR-2018) Synthetic Biology, Wyss Institute, 3
Blackfan Circle, Floor 5, Boston, MA 02115, USA
REFERENCE 3 (bases 1 to 7692)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7692)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Proc. Natl.
Acad. Sci. U.S.A. (2018) In press"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(02-MAR-2018) Synthetic Biology, Wyss Institute, 3 Blackfan Circle,
Floor 5, Boston, MA 02115, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7692
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(157..780)
/label=TetR
/note="tetracycline repressor TetR"
promoter 796..851
/label=tetR/tetA promoters
/note="overlapping promoters for bacterial tetR and tetA"
RBS 869..880
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
CDS 887..4990
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
misc_feature 4991..5020
/label=SGS linker
/note="SGS linker"
CDS 5021..5053
/label=ssrA tag
/note="C-terminal peptide that mediates degradation in
bacteria through the ClpXP and ClpAP proteases (McGinness
et al., 2006)"
misc_feature 5054..5059
/label=double stop
/note="double stop"
terminator 5083..5154
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5170..5197
/label=T7Te terminator
/note="phage T7 early transcription terminator"
misc_RNA 5437..5515
/label=tracrRNA
/note="trans-activating CRISPR RNA for the Streptococcus
pyogenes CRISPR/Cas9 system"
regulatory complement(5553..5594)
/note="derived from Streptococcus pyogenes; tracrRNA
terminator"
/regulatory_class="terminator"
regulatory 5651..5715
/label=J23100 promoter
/note="J23100 promoter"
/regulatory_class="promoter"
repeat_region 5731..5766
/label=DR
/note="direct repeat for the Streptococcus pyogenes
CRISPR/Cas system"
rep_origin 5948..6686
/label=CloDF13 ori
/note="Plasmids containing the CloDF13 (CDF) origin of
replication can be propagated in E. coli cells that contain
additional plasmids with compatible origins."
promoter 6734..6825
/label=AmpR promoter
CDS 6826..7614
/label=SmR
/note="aminoglycoside adenylyltransferase (Murphy, 1985)"
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