Basic Vector Information
Cas9 sgRNA vector 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 ℃.
Cas9 sgRNA vector vector Sequence
LOCUS 40924_430 3951 bp DNA circular SYN 13-MAY-2021 DEFINITION Cas9 sgRNA vector for cloning. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3951) AUTHORS Chen Y, Cao J, Xiong M, Petersen AJ, Dong Y, Tao Y, Huang CT, Du Z, Zhang SC TITLE Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9. JOURNAL Cell Stem Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi: 10.1016/j.stem.2015.06.001. PUBMED 26145478 REFERENCE 2 (bases 1 to 3951) TITLE Direct Submission REFERENCE 3 (bases 1 to 3951) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell Stem Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi: 10.1016/j.stem.2015.06.001." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..3951 /mol_type="other DNA" /organism="synthetic DNA construct" protein_bind 107..128 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /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)." primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" promoter 239..257 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" protein_bind 342..366 /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" promoter complement(460..700) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" promoter complement(840..858) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(865..881) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 1019..1318 /codon_start=1 /label=ccdB /note="CcdB, a bacterial toxin that poisons DNA gyrase" /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" CDS 1670..2461 /codon_start=1 /label=NeoR/KanR /note="aminoglycoside phosphotransferase" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" CDS 2670..3041 /codon_start=1 /label=BleoR /note="antibiotic-binding protein" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" rep_origin 3182..3770 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3924..3941 /label=L4440 /note="L4440 vector, forward primer"
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