LOCUS       pcDNA-dCas9-VP64        9812 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian cell plasmid for CRISPR activation by expressing 
            catalytically inactive dCas9 fused to the VP64 transcriptional 
            activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA-dCas9-VP64
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9812)
  AUTHORS   Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein 
            LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford
            GE, Reddy TE, Gersbach CA.
  TITLE     RNA-guided gene activation by CRISPR-Cas9-based transcription 
            factors.
  JOURNAL   Nat. Methods 2013;10:973-6.
  PUBMED    23892895
REFERENCE   2  (bases 1 to 9812)
  AUTHORS   Gersbach Lab / Addgene #47107
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9812)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Methods"; date: "2013"; volume: "10"; pages: "973-6"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             904..906
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             907..972
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
     CDS             979..999
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             1009..5112
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             5137..5157
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             5167..5316
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=tetrameric repeat of the minimal activation
                     dom
                     /note="VP64"
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             5323..5349
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     polyA_signal    5412..5636
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5682..6110
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6124..6453
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             6520..7311
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    7488..7621
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7658..7674)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7682..7698)
                     /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(7706..7736)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7751..7772)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(8060..8645)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8819..9676)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(9677..9781)
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
      961 gacgatgaca agatggcccc caagaagaag aggaaggtgg gccgcggaat ggataagaaa
     1021 tactcaatag gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
     1081 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
     1141 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
     1201 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
     1261 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
     1321 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
     1381 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
     1441 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
     1501 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
     1561 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt
     1621 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
     1681 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
     1741 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
     1801 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
     1861 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
     1921 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
     1981 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
     2041 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
     2101 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
     2161 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
     2221 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
     2281 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
     2341 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
     2401 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
     2461 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat
     2521 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg
     2581 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag
     2641 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta
     2701 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa
     2761 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa
     2821 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc
     2881 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat
     2941 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga
     3001 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa
     3061 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt
     3121 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt
     3181 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt
     3241 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca
     3301 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc
     3361 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact
     3421 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg
     3481 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatgccat tgttccacaa
     3541 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt
     3601 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga
     3661 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa
     3721 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact
     3781 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat
     3841 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct
     3901 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc
     3961 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt
     4021 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag
     4081 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac
     4141 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa
     4201 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc
     4261 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga
     4321 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa
     4381 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta
     4441 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta
     4501 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct
     4561 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt
     4621 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat
     4681 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag
     4741 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat
     4801 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat
     4861 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa
     4921 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt
     4981 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat
     5041 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag
     5101 ctaggaggtg acccaattgc cggatccaag gctagcccga aaaagaaacg caaagttggg
     5161 cgcgccgacg cgctggacga tttcgatctc gacatgctgg gttctgatgc cctcgatgac
     5221 tttgacctgg atatgttggg aagcgacgca ttggatgact ttgatctgga catgctcggc
     5281 tccgatgctc tggacgattt cgatctcgat atgttaatta actacccgta cgacgttccg
     5341 gactacgctt cttgaatcgg taggaattcg cggccgtcta gacttaagtt taaaccgctg
     5401 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     5461 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     5521 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     5581 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     5641 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     5701 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5761 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5821 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5881 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5941 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     6001 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     6061 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     6121 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     6181 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     6241 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     6301 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     6361 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     6421 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     6481 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     6541 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     6601 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     6661 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     6721 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     6781 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     6841 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     6901 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     6961 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     7021 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     7081 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     7141 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     7201 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     7261 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     7321 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     7381 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     7441 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     7501 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     7561 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     7621 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     7681 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     7741 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     7801 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     7861 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     7921 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     7981 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     8041 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     8101 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     8161 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     8221 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     8281 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     8341 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     8401 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     8461 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     8521 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     8581 ccaccgctgg tagcggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     8641 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     8701 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     8761 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     8821 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     8881 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     8941 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     9001 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     9061 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     9121 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     9181 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     9241 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     9301 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     9361 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     9421 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     9481 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     9541 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     9601 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     9661 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     9721 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     9781 gcacatttcc ccgaaaagtg ccacctgacg tc
//
