Basic Vector Information
MSP2133 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 ℃.
MSP2133 vector Sequence
LOCUS MSP2133. 7614 bp DNA circular SYN 01-JAN-1980 DEFINITION Mammalian cell plasmid for expressing the high-fidelity S. pyogenes Cas9 variant SpCas9-HF4. ACCESSION . VERSION . KEYWORDS MSP2133. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7614) AUTHORS Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK. TITLE High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. JOURNAL Nature 2016;529:490-5. PUBMED 26735016 REFERENCE 2 (bases 1 to 7614) AUTHORS Joung Lab / Addgene #72249 TITLE Direct Submission REFERENCE 3 (bases 1 to 7614) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature"; date: "2016"; volume: "529"; pages: "490-5" 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=SpCas9-HF4 /note="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Kleinstiver et al., 2016)" 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"
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