Basic Vector Information
3xFLAG-dCas9/pMXs-puro 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 ℃.
3xFLAG-dCas9/pMXs-puro vector Sequence
LOCUS 3xFLAG-dCas9_pMX 10003 bp DNA circular SYN 01-JAN-1980 DEFINITION Retroviral vector with a puromycin resistance marker, encoding FLAG(R)-tagged dCas9 for engineered ChIP (enChIP) purification of specific genomic regions. ACCESSION . VERSION . KEYWORDS 3xFLAG-dCas9 pMXs-puro. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10003) AUTHORS Fujita T, Fujii H. TITLE Identification of proteins associated with an IFN-gamma-responsive promoter by a retroviral expression system for enChIP using CRISPR. JOURNAL PLoS ONE 2014;9:e103084. PUBMED 25051498 REFERENCE 2 (bases 1 to 10003) AUTHORS Fujii Lab / Addgene #51240 TITLE Direct Submission REFERENCE 3 (bases 1 to 10003) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "PLoS ONE"; date: "2014"; volume: "9"; pages: "e103084" COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..10003 /mol_type="other DNA" /organism="synthetic DNA construct" LTR 22..615 /note="long terminal repeat from Moloney murine leukemia virus" misc_feature 678..1035 /label=MMLV Psi /note="packaging signal of Moloney murine leukemia virus (MMLV)" CDS 1094..1510 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" misc_feature 1520..1894 /label=pol region /note="Moloney murine leukemia virus (MMLV) pol region containing the splice acceptor site" CDS 1948..1950 /codon_start=1 /product="start codon" /label=start codon /note="ATG" /translation="M" CDS 1951..2016 /label=3xFLAG /note="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" CDS 2020..6123 /label=dCas9 /note="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" CDS 6136..6156 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" promoter 6343..6672 /label=SV40 promoter /note="SV40 enhancer and early promoter" CDS 6682..7278 /label=PuroR /note="puromycin N-acetyltransferase" LTR 7386..7977 /label=LTR /note="long terminal repeat from Moloney murine leukemia virus" rep_origin complement(8278..8866) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(9033..9890) /label=AmpR /note="beta-lactamase" promoter complement(9891..9995) /label=AmpR promoter
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