LOCUS       pAC153-dual-dCas        8703 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 VP96 
            transcriptional activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    pAC153-dual-dCas9VP96-sgExpression
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8703)
  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 8703)
  AUTHORS   Jaenisch Lab / Addgene #48239
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8703)
  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..8703
                     /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..5699
                     /label=VP96
                     /note="6 tandem repeats of the minimal activation domain of
                     herpes simplex virus VP16 (Cheng et al., 2013)"
     polyA_signal    5750..5957
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5966..6106
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6181..6636
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6918..7022
                     /label=AmpR promoter
     CDS             7023..7880
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      8054..8642
                     /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 tttcgatttg gatatgttgt
     5701 atatcgattg attaattaag aattcctaga gctcgctgat cagcctcgac tgtgccttct
     5761 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     5821 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     5881 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     5941 agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     6001 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     6061 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     6121 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     6181 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     6241 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     6301 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     6361 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     6421 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     6481 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     6541 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     6601 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     6661 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     6721 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     6781 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     6841 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     6901 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     6961 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     7021 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     7081 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     7141 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     7201 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     7261 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     7321 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     7381 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     7441 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     7501 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     7561 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     7621 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     7681 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
     7741 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     7801 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     7861 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     7921 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     7981 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     8041 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     8101 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     8161 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     8221 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     8281 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     8341 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     8401 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     8461 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     8521 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     8581 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     8641 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     8701 tgt
//
