LOCUS       sequence-209647-        7231 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses MPH co-activator from the CAG promoter and one U6-driven 
            dgRNA in AAV backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7231)
  AUTHORS   Liao HK, Hatanaka F, Araoka T, Reddy P, Wu MZ, Sui Y, Yamauchi T, 
            Sakurai M, O'Keefe DD, Nunez-Delicado E, Guillen P, Campistol JM, Wu
            CJ, Lu LF, Esteban CR, Izpisua Belmonte JC
  TITLE     In Vivo Target Gene Activation via CRISPR/Cas9-Mediated 
            Trans-epigenetic Modulation.
  JOURNAL   Cell. 2017 Dec 14;171(7):1495-1507.e15. doi: 
            10.1016/j.cell.2017.10.025. Epub 2017 Dec 7.
  PUBMED    29224783
REFERENCE   2  (bases 1 to 7231)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2017.10.025"; journalName: "Cell"; date: 
            "2017-12-14- 14"; volume: "171"; issue: "7"; pages: "1495-1507.e15"
COMMENT     Sequence Label: AAV-U6-dgRNA-CAG-MPH
FEATURES             Location/Qualifiers
     source          1..7231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        149..397
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     149..169
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     320..339
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        632..935
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        937..1213
                     /label=chicken beta-actin promoter
     intron          1214..2230
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2154..2176
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2238..2257
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    2285..2309
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2331..2717
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             2772..2792
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2808..3350
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             3375..3746
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     protein_bind    complement(3780..3804)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3836..4424
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3889..3909)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4307..4318)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4438..4486
                     /note="synthetic polyadenylation signal"
     repeat_region   4494..4634
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4494..4623
                     /label=AAV2 ITR
     rep_origin      4709..5164
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4796..4815)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5006..5027
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5181..5200)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5300..5322
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5360..5378)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5446..5550
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5551..5619,5620..6411)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5769..5788)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6582..7170
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7071..7090
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gaacgcgtga gggcctattt cccatgattc cttcatattt
      181 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
      241 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
      301 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
      361 cttggcttta tatatcttgt ggaaaggacg aaacaccgga agagcgagct cttctgtttc
      421 agagctaggc cagcatgagg atcacccatg cctgcagggc ctagcaagtt gaaataaggc
      481 tagtccgtta tcaacttggc cagcatgagg atcacccatg cctgcagggc caagtggcac
      541 cgagtcggtg cttttttgca tatgctagtg cgcgttatta atagtaatca attacggggt
      601 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      661 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      721 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      781 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      841 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      901 agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct
      961 tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt attttttaat
     1021 tattttgtgc agcgatgggg gcgggggggg ggggggggcg cgcgccaggc ggggcggggc
     1081 ggggcgaggg gcggggcggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
     1141 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
     1201 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
     1261 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
     1321 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct
     1381 gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc
     1441 ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg
     1501 cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg
     1561 gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg
     1621 cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca
     1681 accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct
     1741 ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt
     1801 gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc
     1861 ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc
     1921 gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag
     1981 gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga
     2041 aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc
     2101 tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg
     2161 cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt
     2221 tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga
     2281 attcacaagt ttgtacaaaa aagcaggctc cgaattcacc ggtcaccatg gcttcaaact
     2341 ttactcagtt cgtgctcgtg gacaatggtg ggacagggga tgtgacagtg gctccttcta
     2401 atttcgctaa tggggtggca gagtggatca gctccaactc acggagccag gcctacaagg
     2461 tgacatgcag cgtcaggcag tctagtgccc agaagagaaa gtataccatc aaggtggagg
     2521 tccccaaagt ggctacccag acagtgggcg gagtcgaact gcctgtcgcc gcttggaggt
     2581 cctacctgaa catggagctc actatcccaa ttttcgctac caattctgac tgtgaactca
     2641 tcgtgaaggc aatgcagggg ctcctcaaag acggtaatcc tatcccttcc gccatcgccg
     2701 ctaactcagg tatctacagc gctggaggag gtggaagcgg aggaggagga agcggaggag
     2761 gaggtagcgg acctaagaaa aagaggaagg tggcggccgc tggatcccct tcagggcaga
     2821 tcagcaacca ggccctggct ctggccccta gctccgctcc agtgctggcc cagactatgg
     2881 tgccctctag tgctatggtg cctctggccc agccacctgc tccagcccct gtgctgaccc
     2941 caggaccacc ccagtcactg agcgctccag tgcccaagtc tacacaggcc ggcgagggga
     3001 ctctgagtga agctctgctg cacctgcagt tcgacgctga tgaggacctg ggagctctgc
     3061 tggggaacag caccgatccc ggagtgttca cagatctggc ctccgtggac aactctgagt
     3121 ttcagcagct gctgaatcag ggcgtgtcca tgtctcatag tacagccgaa ccaatgctga
     3181 tggagtaccc cgaagccatt acccggctgg tgaccggcag ccagcggccc cccgaccccg
     3241 ctccaactcc cctgggaacc agcggcctgc ctaatgggct gtccggagat gaagacttct
     3301 caagcatcgc tgatatggac tttagtgccc tgctgtcaca gatttcctct agtgggcagg
     3361 gaggaggtgg aagcggcttc agcgtggaca ccagtgccct gctggacctg ttcagcccct
     3421 cggtgaccgt gcccgacatg agcctgcctg accttgacag cagcctggcc agtatccaag
     3481 agctcctgtc tccccaggag ccccccaggc ctcccgaggc agagaacagc agcccggatt
     3541 cagggaagca gctggtgcac tacacagcgc agccgctgtt cctgctggac cccggctccg
     3601 tggacaccgg gagcaacgac ctgccggtgc tgtttgagct gggagagggc tcctacttct
     3661 ccgaagggga cggcttcgcc gaggacccca ccatctccct gctgacaggc tcggagcctc
     3721 ccaaagccaa ggaccccact gtctccgcta gcggcatcga ttgattaatt aagaattcga
     3781 cccagctttc ttgtacaaag tggtgaattc aagcttcggg gactagtcat atgataatca
     3841 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     3901 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     3961 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     4021 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     4081 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     4141 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     4201 cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg
     4261 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     4321 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     4381 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcacaatt ggaattcaat
     4441 aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgcggc cgcaggaacc
     4501 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4561 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     4621 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4681 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     4741 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     4801 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4861 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4921 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4981 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5041 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     5101 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     5161 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     5221 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5281 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5341 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     5401 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5461 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5521 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5581 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5641 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5701 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5761 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5821 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5881 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5941 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6001 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6061 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6121 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6181 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6241 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     6301 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6361 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6421 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6481 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6541 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6601 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6661 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6721 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6781 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6841 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6901 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6961 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7021 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7081 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7141 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7201 ctggcctttt gctggccttt tgctcacatg t
//
