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LOCUS       3xFLAG-dCas9_pCM        8848 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for expression in mammalian cells of FLAG(R)-tagged 
            catalytically inactive dCas9, for engineered ChIP (enChIP) 
            purification of specific genomic regions.
ACCESSION   .
VERSION     .
KEYWORDS    3xFLAG-dCas9 pCMV-7.1.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8848)
  AUTHORS   Fujita T, Fujii H.
  TITLE     Efficient isolation of specific genomic regions and identification 
            of associated proteins by engineered DNA-binding molecule-mediated 
            chromatin immunoprecipitation (enChIP) using CRISPR.
  JOURNAL   Biochem. Biophys. Res. Commun. 2013;439:132-6.
  PUBMED    23942116
REFERENCE   2  (bases 1 to 8848)
  AUTHORS   Fujii Lab / Addgene #47948
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8848)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biochem. 
            Biophys. Res. Commun."; date: "2013"; volume: "439"; pages: "132-6"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8848
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             928..930
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             931..996
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
     CDS             1000..5103
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             5116..5136
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     polyA_signal    5189..5811
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     promoter        5840..6169
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        complement(6208..6226)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6240..6256)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(6264..6280)
                     /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(6288..6318)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6333..6354)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6642..7230)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7404..8261)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8262..8366)
                     /label=AmpR promoter
     rep_origin      8393..8848
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"