LOCUS       pcDNA-dCas9.        9575 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for expressing catalytically inactive dCas9 in mammalian 
            cells using the CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA-dCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9575)
  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 9575)
  AUTHORS   Gersbach Lab / Addgene #47106
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9575)
  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..9575
                     /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
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=three tandem FLAG
                     /note="3xFLAG"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             979..999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             1009..5112
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             5113..5133
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     polyA_signal    5175..5399
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5445..5873
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5887..6216
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             6283..7074
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    7251..7384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7421..7437)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7445..7461)
                     /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(7469..7499)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(7514..7535)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(7823..8408)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8582..9439)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(9440..9544)
                     /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 accccaagaa gaagaggaag gtgggctgat ctagacttaa gtttaaaccg
     5161 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     5221 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     5281 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     5341 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     5401 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     5461 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5521 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5581 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5641 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5701 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5761 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     5821 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     5881 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     5941 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     6001 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     6061 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     6121 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     6181 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     6241 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     6301 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     6361 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     6421 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     6481 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     6541 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     6601 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     6661 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     6721 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     6781 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     6841 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     6901 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     6961 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     7021 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     7081 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     7141 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     7201 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     7261 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     7321 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     7381 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     7441 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     7501 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     7561 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     7621 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     7681 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     7741 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     7801 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     7861 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     7921 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     7981 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     8041 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     8101 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     8161 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     8221 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     8281 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     8341 aaaccaccgc tggtagcggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     8401 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     8461 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     8521 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     8581 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     8641 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     8701 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     8761 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     8821 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     8881 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     8941 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     9001 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     9061 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     9121 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     9181 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     9241 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     9301 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     9361 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     9421 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     9481 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     9541 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
