LOCUS       pAC154-dual-dCas        8859 bp DNA     circular SYN 01-JAN-1980
DEFINITION  CRISPR activation vector for co-expressing a single guide RNA 
            (sgRNA) with catalytically inactive dCas9 fused to the VP160 
            transcriptional activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    pAC154-dual-dCas9VP160-sgExpression
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8859)
  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 8859)
  AUTHORS   Jaenisch Lab / Addgene #48240
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8859)
  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..8859
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      344..349
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1233
                     /note="hybrid intron"
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1251..1253
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             1254..1280
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="HA"
                     /translation="YPYDVPDYA"
     CDS             1284..1304
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             1314..5414
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             5418..5438
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             5472..5855
                     /label=VP160
                     /note="10 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
     polyA_signal    5906..6113
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6122..6262
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6337..6792
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        7074..7178
                     /label=AmpR promoter
     CDS             7179..8036
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      8210..8798
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atgtacccat
     1261 acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg tccgacaaga
     1321 agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg
     1381 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1441 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1501 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1561 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1621 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1681 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1741 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1801 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1861 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1921 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     1981 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac ctgattgccc
     2041 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     2101 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     2161 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     2221 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     2281 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     2341 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     2401 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2461 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2521 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2581 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2641 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2701 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2761 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2821 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2881 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     2941 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     3001 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     3061 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     3121 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     3181 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     3241 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     3301 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     3361 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     3421 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3481 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3541 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3601 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3661 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3721 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3781 tggaccagga actggacatc aaccggctgt ccgactacga tgtggacgcc atcgtgcctc
     3841 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3901 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     3961 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     4021 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     4081 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     4141 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     4201 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     4261 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     4321 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     4381 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4441 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4501 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4561 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4621 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4681 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4741 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4801 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4861 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4921 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     4981 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     5041 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     5101 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     5161 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     5221 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     5281 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     5341 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     5401 agctgggagg cgacagcccc aagaagaaga gaaaggtgga ggccagcggg ccggccggat
     5461 ccgggcgcgc cgacgcgctg gacgatttcg atctcgacat gctgggttct gatgccctcg
     5521 atgactttga cctggatatg ttgggaagcg acgcattgga tgactttgat ctggacatgc
     5581 tcggctccga tgctctggac gatttcgatc tcgatatgtt agggtcagac gcactggatg
     5641 atttcgacct tgatatgttg ggaagcgatg cccttgatga tttcgacctg gacatgctcg
     5701 gcagcgacgc cctggacgat ttcgatctgg acatgctggg gtccgatgcc ttggatgatt
     5761 ttgacttgga tatgctgggg agtgatgccc tggacgactt tgacctggac atgctgggct
     5821 ccgatgcgct cgatgacttc gatttggata tgttgtatat cgattgatta attaagaatt
     5881 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     5941 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     6001 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     6061 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg
     6121 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     6181 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     6241 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     6301 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     6361 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     6421 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     6481 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     6541 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     6601 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     6661 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     6721 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     6781 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6841 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     6901 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     6961 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     7021 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     7081 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     7141 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     7201 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     7261 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     7321 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     7381 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     7441 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     7501 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     7561 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     7621 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     7681 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     7741 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     7801 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7861 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     7921 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7981 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     8041 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     8101 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     8161 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     8221 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     8281 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     8341 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     8401 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     8461 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     8521 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     8581 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     8641 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     8701 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     8761 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     8821 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
