Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012319 | MSP1101 | 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:
- MSP1101
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7614 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z,
- Copy Number:
- High copy number
MSP1101 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.
MSP1101 vector Sequence
LOCUS MSP1101. 7614 bp DNA circular SYN 01-JAN-1980
DEFINITION Vector for expressing the S. pyogenes Cas9 VRER mutant, which has an
altered PAM recognition specificity.
ACCESSION .
VERSION .
KEYWORDS MSP1101
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7614)
AUTHORS Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z,
Gonzales AP, Li Z, Peterson RT, Yeh JR, Aryee MJ, Joung JK.
TITLE Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
JOURNAL Nature 2015;523:481-5.
PUBMED 26098369
REFERENCE 2 (bases 1 to 7614)
AUTHORS Joung Lab / Addgene #65773
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7614)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature";
date: "2015"; volume: "523"; pages: "481-5"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7614
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 132..335
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 377..395
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 409..4512
/label=Cas9 VRER
/note="Streptococcus pyogenes Cas9 endonuclease with the
D1135V/G1218R/R1335E/T1337R mutations"
CDS 4519..4539
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 4546..4611
/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 4629..4646
/label=6xHis
/note="6xHis affinity tag"
polyA_signal 4675..4899
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(4970..4986)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(4994..5010)
/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(5018..5048)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(5063..5084)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(5372..5960)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6134..6991)
/label=AmpR
/note="beta-lactamase"
promoter complement(6992..7096)
/label=AmpR promoter
enhancer 7366..7614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"