Basic Vector Information
MSP1101 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 ℃.
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"
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