Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012346 | eSpCas9(1.1) | 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:
- eSpCas9(1.1)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8506 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F.
- Copy Number:
- High copy number
- Promoter:
- CBh
eSpCas9(1.1) 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.
eSpCas9(1.1) vector Sequence
LOCUS eSpCas9(1.1). 8506 bp DNA circular SYN 01-JAN-1980
DEFINITION Zhang lab vector for expressing a single guide RNA (sgRNA) together
with an S. pyogenes Cas9 variant with improved targeting
specificity.
ACCESSION .
VERSION .
KEYWORDS eSpCas9(1.1)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8506)
AUTHORS Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F.
TITLE Rationally engineered Cas9 nucleases with improved specificity.
JOURNAL Science 2016;351:84-8.
PUBMED 26628643
REFERENCE 2 (bases 1 to 8506)
AUTHORS Zhang Lab / Addgene #71814
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8506)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Science";
date: "2016"; volume: "351"; pages: "84-8"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT An error in codon 848 was corrected based on sequencing results from
Addgene.
FEATURES Location/Qualifiers
source 1..8506
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..241
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 268..343
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator 344..349
/note="polIII terminator"
/note="RNA polymerase III transcription terminator"
enhancer 440..725
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 727..1004
/label=chicken beta-actin promoter
intron 1005..1233
/note="hybrid intron"
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 1251..1253
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 1254..1319
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=three tandem FLAG
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1326..1346
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1371..5471
/label=eSpCas9(1.1)
/note="Cas9 endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system, mutated to improve targeting
specificity (Slaymaker et al., 2016)"
CDS 5472..5519
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=bipartite nuclear localization signal from
nucl
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
polyA_signal 5553..5760
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 5769..5909
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 5984..6439
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6721..6825
/label=AmpR promoter
CDS 6826..7683
/label=AmpR
/note="beta-lactamase"
rep_origin 7857..8445
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"