Basic Vector Information
pmU6-gRNA 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 ℃.
pmU6-gRNA vector Sequence
LOCUS 40924_32703 3542 bp DNA circular SYN 13-MAY-2021 DEFINITION Expresses the S. pyogenes sgRNA from the mouse U6 promoter. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3542) AUTHORS Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA TITLE Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. JOURNAL Nucleic Acids Res. 2014 Aug 13. pii: gku749. PUBMED 25122746 REFERENCE 2 (bases 1 to 3542) TITLE Direct Submission REFERENCE 3 (bases 1 to 3542) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic Acids Res. 2014 Aug 13. pii: gku749." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..3542 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 1..314 /label=U6 promoter /note="RNA polymerase III promoter for mouse U6 snRNA (Das et al., 1988)" misc_RNA 332..407 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(452..470) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(491..507) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(515..531) /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(539..569) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(584..605) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(722..739) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(893..1481) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(1561..2352) /codon_start=1 /label=NeoR/KanR /note="aminoglycoside phosphotransferase" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" promoter complement(2387..2744) /label=SV40 promoter /note="SV40 enhancer and early promoter" promoter complement(2746..2850) /label=AmpR promoter rep_origin complement(2876..3331) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 3473..3489 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 3499..3517 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase"
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