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"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attaaccatg gactacaaag accatgacgg tgattataaa
      961 gatcatgaca tcgattacaa ggatgacgat gacaagctca tggacaagaa gtactccatt
     1021 gggctcgcta tcggcacaaa cagcgtcggc tgggccgtca ttacggacga gtacaaggtg
     1081 ccgagcaaaa aattcaaagt tctgggcaat accgatcgcc acagcataaa gaagaacctc
     1141 attggcgccc tcctgttcga ctccggggag acggccgaag ccacgcggct caaaagaaca
     1201 gcacggcgca gatatacccg cagaaagaat cggatctgct acctgcagga gatctttagt
     1261 aatgagatgg ctaaggtgga tgactctttc ttccataggc tggaggagtc ctttttggtg
     1321 gaggaggata aaaagcacga gcgccaccca atctttggca atatcgtgga cgaggtggcg
     1381 taccatgaaa agtacccaac catatatcat ctgaggaaga agcttgtaga cagtactgat
     1441 aaggctgact tgcggttgat ctatctcgcg ctggcgcata tgatcaaatt tcggggacac
     1501 ttcctcatcg agggggacct gaacccagac aacagcgatg tcgacaaact ctttatccaa
     1561 ctggttcaga cttacaatca gcttttcgaa gagaacccga tcaacgcatc cggagttgac
     1621 gccaaagcaa tcctgagcgc taggctgtcc aaatcccggc ggctcgaaaa cctcatcgca
     1681 cagctccctg gggagaagaa gaacggcctg tttggtaatc ttatcgccct gtcactcggg
     1741 ctgaccccca actttaaatc taacttcgac ctggccgaag atgccaagct tcaactgagc
     1801 aaagacacct acgatgatga tctcgacaat ctgctggccc agatcggcga ccagtacgca
     1861 gacctttttt tggcggcaaa gaacctgtca gacgccattc tgctgagtga tattctgcga
     1921 gtgaacacgg agatcaccaa agctccgctg agcgctagta tgatcaagcg ctatgatgag
     1981 caccaccaag acttgacttt gctgaaggcc cttgtcagac agcaactgcc tgagaagtac
     2041 aaggaaattt tcttcgatca gtctaaaaat ggctacgccg gatacattga cggcggagca
     2101 agccaggagg aattttacaa atttattaag cccatcttgg aaaaaatgga cggcaccgag
     2161 gagctgctgg taaagcttaa cagagaagat ctgttgcgca aacagcgcac tttcgacaat
     2221 ggaagcatcc cccaccagat tcacctgggc gaactgcacg ctatcctcag gcggcaagag
     2281 gatttctacc cctttttgaa agataacagg gaaaagattg agaaaatcct cacatttcgg
     2341 ataccctact atgtaggccc cctcgcccgg ggaaattcca gattcgcgtg gatgactcgc
     2401 aaatcagaag agaccatcac tccctggaac ttcgaggaag tcgtggataa gggggcctct
     2461 gcccagtcct tcatcgaaag gatgactaac tttgataaaa atctgcctaa cgaaaaggtg
     2521 cttcctaaac actctctgct gtacgagtac ttcacagttt ataacgagct caccaaggtc
     2581 aaatacgtca cagaagggat gagaaagcca gcattcctgt ctggagagca gaagaaagct
     2641 atcgtggacc tcctcttcaa gacgaaccgg aaagttaccg tgaaacagct caaagaagac
     2701 tatttcaaaa agattgaatg tttcgactct gttgaaatca gcggagtgga ggatcgcttc
     2761 aacgcatccc tgggaacgta tcacgatctc ctgaaaatca ttaaagacaa ggacttcctg
     2821 gacaatgagg agaacgagga cattcttgag gacattgtcc tcacccttac gttgtttgaa
     2881 gatagggaga tgattgaaga acgcttgaaa acttacgctc atctcttcga cgacaaagtc
     2941 atgaaacagc tcaagaggcg ccgatataca ggatgggggc ggctgtcaag aaaactgatc
     3001 aatgggatcc gagacaagca gagtggaaag acaatcctgg attttcttaa gtccgatgga
     3061 tttgccaaca ggaacttcat gcagttgatc catgatgact ctctcacctt taaggaggac
     3121 atccagaaag cacaagtttc tggccagggg gacagtcttc acgagcacat cgctaatctt
     3181 gcaggtagcc cagctatcaa aaagggaata ctgcagaccg ttaaggtcgt ggatgaactc
     3241 gtcaaagtaa tgggaaggca taagcccgag aatatcgtta tcgagatggc ccgagagaac
     3301 caaactaccc agaagggaca gaagaacagt agggaaagga tgaagaggat tgaagagggt
     3361 ataaaagaac tggggtccca aatccttaag gaacacccag ttgaaaacac ccagcttcag
     3421 aatgagaagc tctacctgta ctacctgcag aacggcaggg acatgtacgt ggatcaggaa
     3481 ctggacatca atcggctctc cgactacgac gtggatgcca tcgtgcccca gtcttttctc
     3541 aaagatgatt ctattgataa taaagtgttg acaagatccg ataaaaatag agggaagagt
     3601 gataacgtcc cctcagaaga agttgtcaag aaaatgaaaa attattggcg gcagctgctg
     3661 aacgccaaac tgatcacaca acggaagttc gataatctga ctaaggctga acgaggtggc
     3721 ctgtctgagt tggataaagc cggcttcatc aaaaggcagc ttgttgagac acgccagatc
     3781 accaagcacg tggcccaaat tctcgattca cgcatgaaca ccaagtacga tgaaaatgac
     3841 aaactgattc gagaggtgaa agttattact ctgaagtcta agctggtctc agatttcaga
     3901 aaggactttc agttttataa ggtgagagag atcaacaatt accaccatgc gcatgatgcc
     3961 tacctgaatg cagtggtagg cactgcactt atcaaaaaat atcccaagct tgaatctgaa
     4021 tttgtttacg gagactataa agtgtacgat gttaggaaaa tgatcgcaaa gtctgagcag
     4081 gaaataggca aggccaccgc taagtacttc ttttacagca atattatgaa ttttttcaag
     4141 accgagatta cactggccaa tggagagatt cggaagcgac cacttatcga aacaaacgga
     4201 gaaacaggag aaatcgtgtg ggacaagggt agggatttcg cgacagtccg gaaggtcctg
     4261 tccatgccgc aggtgaacat cgttaaaaag accgaagtac agaccggagg cttctccaag
     4321 gaaagtatcc tcccgaaaag gaacagcgac aagctgatcg cacgcaaaaa agattgggac
     4381 cccaagaaat acggcggatt cgattctcct acagtcgctt acagtgtact ggttgtggcc
     4441 aaagtggaga aagggaagtc taaaaaactc aaaagcgtca aggaactgct gggcatcaca
     4501 atcatggagc gatcaagctt cgaaaaaaac cccatcgact ttctcgaggc gaaaggatat
     4561 aaagaggtca aaaaagacct catcattaag cttcccaagt actctctctt tgagcttgaa
     4621 aacggccgga aacgaatgct cgctagtgcg ggcgagctgc agaaaggtaa cgagctggca
     4681 ctgccctcta aatacgttaa tttcttgtat ctggccagcc actatgaaaa gctcaaaggg
     4741 tctcccgaag ataatgagca gaagcagctg ttcgtggaac aacacaaaca ctaccttgat
     4801 gagatcatcg agcaaataag cgaattctcc aaaagagtga tcctcgccga cgctaacctc
     4861 gataaggtgc tttctgctta caataagcac agggataagc ccatcaggga gcaggcagaa
     4921 aacattatcc acttgtttac tctgaccaac ttgggcgcgc ctgcagcctt caagtacttc
     4981 gacaccacca tagacagaaa gcggtacacc tctacaaagg aggtcctgga cgccacactg
     5041 attcatcagt caattacggg gctctatgaa acaagaatcg acctctctca gctcggtgga
     5101 gacagcaggg ctgaccccaa gaagaagagg aaggtgtgaa agggttcgat ccctaccgga
     5161 tcggtaccag tcgactctag aggatcccgg gtggcatccc tgtgacccct ccccagtgcc
     5221 tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag
     5281 ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt
     5341 atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc
     5401 aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc
     5461 gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag
     5521 ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa
     5581 ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg
     5641 aaccactgct cccttccctg tccttctgat tttaaaataa ctataccagc aggaggacgt
     5701 ccagacacag cataggctac ctggccatgc ccaaccggtg ggacatttga gttgcttgct
     5761 tggcactgtc ctctcatgcg ttgggtccac tcagtagatg cctgttgaat tgggtacgcg
     5821 gccagcttgg ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     5881 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     5941 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     6001 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     6061 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     6121 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcctcga
     6181 ggaactgaaa aaccagaaag ttaattccct atagtgagtc gtattaaatt cgtaatcatg
     6241 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     6301 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     6361 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6421 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     6481 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     6541 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     6601 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     6661 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     6721 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6781 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6841 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6901 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6961 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     7021 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     7081 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     7141 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     7201 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     7261 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     7321 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     7381 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     7441 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     7501 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     7561 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     7621 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     7681 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     7741 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     7801 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7861 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7921 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7981 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     8041 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     8101 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     8161 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     8221 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     8281 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     8341 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc
     8401 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     8461 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     8521 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     8581 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     8641 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     8701 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     8761 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     8821 tttaacaaaa tattaacgct tacaattt
//
