LOCUS       pAC94-pmax-dCas9        8282 bp DNA     circular SYN 01-JAN-1980
DEFINITION  CRISPR activation vector for expressing a puromycin resistance 
            marker plus catalytically inactive dCas9 fused to the VP160 
            transcriptional activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    pAC94-pmax-dCas9VP160-2A-puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8282)
  AUTHORS   Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan 
            S, Shivalila CS, Dadon DB, Jaenisch R.
  TITLE     Multiplexed activation of endogenous genes by CRISPR-on, an 
            RNA-guided transcriptional activator system.
  JOURNAL   Cell Res. 2013;23:1163-71.
  PUBMED    23979020
REFERENCE   2  (bases 1 to 8282)
  AUTHORS   Jaenisch Lab / Addgene #48226
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8282)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Res.";
            date: "2013"; volume: "23"; pages: "1163-71"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        139..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..719
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     intron          813..945
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     protein_bind    960..984
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1005..1007
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             1008..1034
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="HA"
                     /translation="YPYDVPDYA"
     CDS             1038..1058
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             1068..5168
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             5172..5192
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             5226..5609
                     /label=VP160
                     /note="10 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
     CDS             5634..5687
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
     CDS             5697..6293
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
     protein_bind    complement(6312..6336)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6389..6430
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
     polyA_signal    complement(6485..6566)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(6764..7351)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7435..8226)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg
      721 tcgtttagtg aaccgtcaga tcactagtag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctc gactgatcac aggtaagtat caaggttaca agacaggttt
      841 aaggaggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg
      901 cacctattgg tcttactgac atccactttg cctttctctc cacagggaaa aaacaattga
      961 caagtttgta caaaaaagca ggctccgaat tcaccggtgc caccatgtac ccatacgatg
     1021 ttccagatta cgcttcgccg aagaaaaagc gcaaggtcga agcgtccgac aagaagtaca
     1081 gcatcggcct ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca
     1141 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
     1201 acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
     1261 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct
     1321 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc
     1381 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg
     1441 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca
     1501 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg
     1561 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
     1621 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg
     1681 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga
     1741 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc
     1801 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc
     1861 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
     1921 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc
     1981 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg
     2041 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga
     2101 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg
     2161 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca
     2221 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg
     2281 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc
     2341 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct
     2401 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga
     2461 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg
     2521 cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga
     2581 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca
     2641 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
     2701 aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag
     2761 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc
     2821 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact
     2881 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt
     2941 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca
     3001 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc
     3061 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg
     3121 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag
     3181 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca
     3241 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg
     3301 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag
     3361 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag
     3421 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc
     3481 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc
     3541 aggaactgga catcaaccgg ctgtccgact acgatgtgga cgccatcgtg cctcagagct
     3601 ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca
     3661 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc
     3721 tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag
     3781 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc
     3841 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga
     3901 atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt
     3961 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg
     4021 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
     4081 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
     4141 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt
     4201 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa
     4261 acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag
     4321 tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca
     4381 gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact
     4441 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg
     4501 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga
     4561 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg
     4621 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc
     4681 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac
     4741 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga
     4801 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc
     4861 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta
     4921 atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg
     4981 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt
     5041 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca
     5101 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg
     5161 gaggcgacag ccccaagaag aagagaaagg tggaggccag cgggccggcc ggatccgggc
     5221 gcgccgacgc gctggacgat ttcgatctcg acatgctggg ttctgatgcc ctcgatgact
     5281 ttgacctgga tatgttggga agcgacgcat tggatgactt tgatctggac atgctcggct
     5341 ccgatgctct ggacgatttc gatctcgata tgttagggtc agacgcactg gatgatttcg
     5401 accttgatat gttgggaagc gatgcccttg atgatttcga cctggacatg ctcggcagcg
     5461 acgccctgga cgatttcgat ctggacatgc tggggtccga tgccttggat gattttgact
     5521 tggatatgct ggggagtgat gccctggacg actttgacct ggacatgctg ggctccgatg
     5581 cgctcgatga cttcgatttg gatatgttgt atatcgatag atctggcagc ggagagggca
     5641 gaggaagcct tctaacatgc ggtgacgtgg aggagaatcc cggccctagg ctcgagatga
     5701 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     5761 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     5821 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     5881 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     5941 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     6001 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     6061 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     6121 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     6181 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     6241 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgattaa
     6301 ttaagaattc gacccagctt tcttgtacaa agtggttgat atccagcaca gtggcggccg
     6361 ctcgagtcta gagggcccgc ggttcgaagg taagcctatc cctaaccctc tcctcggtct
     6421 cgattctacg cgtaccggtt agcaattgtt ttttcgatga gtttggacaa accacaacta
     6481 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     6541 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     6601 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtactt
     6661 aagaggggga gaccaaaggg cgagacgtta aggcctcacg tgacatgtga gcaaaaggcc
     6721 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6781 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6841 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6901 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6961 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     7021 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     7081 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     7141 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     7201 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     7261 gtagctcttg atccggcaaa caaaccacgc tggtagcggt ggtttttttg tttgcaagca
     7321 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     7381 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgccgt ctcagaagaa
     7441 ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag
     7501 cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa
     7561 cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa
     7621 gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc
     7681 ctcgccgtcg ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg
     7741 atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg
     7801 ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag
     7861 ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag
     7921 gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac
     7981 gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc
     8041 gtcttgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc
     8101 ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc
     8161 atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc
     8221 aatcataata ttattgaagc atttatcagg gttcgtctcg tcccggtctc ctcccatgca
     8281 tg
//
