LOCUS       pAC149-pCR8-dCas        7442 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Gateway(R) entry vector with a cassette encoding catalytically 
            inactive dCas9 fused to the VP160 transcriptional activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    pAC149-pCR8-dCas9VP160.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7442)
  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 7442)
  AUTHORS   Jaenisch Lab / Addgene #48221
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7442)
  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..7442
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(387..473)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /label=attL1
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             689..691
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             692..718
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="HA"
                     /translation="YPYDVPDYA"
     CDS             722..742
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             752..4852
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             4856..4876
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             4910..5293
                     /label=VP160
                     /note="10 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
     protein_bind    complement(5321..5420)
                     /label=attL2
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(5438..5456)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5461..5477)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5908..6696
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     rep_origin      6792..7380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gaattcaccg gtgccaccat gtacccatac gatgttccag attacgcttc
      721 gccgaagaaa aagcgcaagg tcgaagcgtc cgacaagaag tacagcatcg gcctggccat
      781 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
      841 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
      901 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
      961 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     1021 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     1081 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     1141 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     1201 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     1261 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     1321 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     1381 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     1441 cgagaagaag aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa
     1501 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     1561 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     1621 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     1681 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     1741 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     1801 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     1861 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     1921 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     1981 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     2041 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     2101 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     2161 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     2221 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     2281 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     2341 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     2401 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     2461 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     2521 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     2581 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     2641 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     2701 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     2761 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     2821 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     2881 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     2941 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     3001 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     3061 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     3121 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     3181 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     3241 ccggctgtcc gactacgatg tggacgccat cgtgcctcag agctttctga aggacgactc
     3301 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     3361 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     3421 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     3481 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     3541 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     3601 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     3661 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     3721 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     3781 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     3841 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     3901 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     3961 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     4021 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     4081 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     4141 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     4201 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     4261 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     4321 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     4381 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     4441 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     4501 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     4561 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     4621 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     4681 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     4741 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     4801 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acagccccaa
     4861 gaagaagaga aaggtggagg ccagcgggcc ggccggatcc gggcgcgccg acgcgctgga
     4921 cgatttcgat ctcgacatgc tgggttctga tgccctcgat gactttgacc tggatatgtt
     4981 gggaagcgac gcattggatg actttgatct ggacatgctc ggctccgatg ctctggacga
     5041 tttcgatctc gatatgttag ggtcagacgc actggatgat ttcgaccttg atatgttggg
     5101 aagcgatgcc cttgatgatt tcgacctgga catgctcggc agcgacgccc tggacgattt
     5161 cgatctggac atgctggggt ccgatgcctt ggatgatttt gacttggata tgctggggag
     5221 tgatgccctg gacgactttg acctggacat gctgggctcc gatgcgctcg atgacttcga
     5281 tttggatatg ttgtatatcg attgattaat taagaattcg acccagcttt cttgtacaaa
     5341 gttggcatta taaaaaataa ttgctcatca atttgttgca acgaacaggt cactatcagt
     5401 caaaataaaa tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg
     5461 gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca
     5521 caagataaaa atatatcatc atgcctcctc tagaccagcc aggacagaaa tgcctcgact
     5581 tcgctgctgc ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc
     5641 cagtggacat aagcctgttc ggttcgtaag ctgtaatgca agtagcgtat gcgctcacgc
     5701 aactggtcca gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg
     5761 gcttgttatg actgtttttt tggggtacag tctatgcctc gggcatccaa gcagcaagcg
     5821 cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac gcagcagggc
     5881 agtcgcccta aaacaaagtt aaacatcatg agggaagcgg tgatcgccga agtatcgact
     5941 caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta
     6001 catttgtacg gctccgcagt ggatggcggc ctgaagccac acagtgatat tgatttgctg
     6061 gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg
     6121 gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt
     6181 gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa
     6241 tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg
     6301 gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag
     6361 gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg
     6421 ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc
     6481 cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg
     6541 gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat
     6601 cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccac
     6661 tacgtgaaag gcgagatcac caaggtagtc ggcaaataac cctcgagcca cccatgacca
     6721 aaatccctta acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc
     6781 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6841 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6901 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     6961 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     7021 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     7081 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     7141 gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg
     7201 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7261 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7321 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7381 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7441 tt
//
