LOCUS       sequence-209646-        6609 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses MPH co-activator from the CAG promoter and one U6-driven 
            dgRNA in AAV backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6609)
  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 6609)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 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..6609
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     479..501
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     563..582
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    610..634
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             656..1042
                     /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             1097..1117
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1133..1675
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             1700..2071
                     /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(2105..2129)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2161..2749
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2214..2234)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2632..2643)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2763..2811
                     /note="synthetic polyadenylation signal"
     repeat_region   2819..2959
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2819..2948
                     /label=AAV2 ITR
     rep_origin      3034..3489
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3121..3140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3331..3352
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3506..3525)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3625..3647
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3685..3703)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3771..3875
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3876..3944,3945..4736)
                     /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(4094..4113)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4907..5495
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5396..5415
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5557..5686
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        5705..5953
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5705..5725
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5876..5895
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        6188..6491
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ctgcaacccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg
       61 gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg
      121 ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc
      181 ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg
      241 taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct
      301 gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg
      361 aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc
      421 cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg
      481 ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc
      541 ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc
      601 aaagaattca caagtttgta caaaaaagca ggctccgaat tcaccggtca ccatggcttc
      661 aaactttact cagttcgtgc tcgtggacaa tggtgggaca ggggatgtga cagtggctcc
      721 ttctaatttc gctaatgggg tggcagagtg gatcagctcc aactcacgga gccaggccta
      781 caaggtgaca tgcagcgtca ggcagtctag tgcccagaag agaaagtata ccatcaaggt
      841 ggaggtcccc aaagtggcta cccagacagt gggcggagtc gaactgcctg tcgccgcttg
      901 gaggtcctac ctgaacatgg agctcactat cccaattttc gctaccaatt ctgactgtga
      961 actcatcgtg aaggcaatgc aggggctcct caaagacggt aatcctatcc cttccgccat
     1021 cgccgctaac tcaggtatct acagcgctgg aggaggtgga agcggaggag gaggaagcgg
     1081 aggaggaggt agcggaccta agaaaaagag gaaggtggcg gccgctggat ccccttcagg
     1141 gcagatcagc aaccaggccc tggctctggc ccctagctcc gctccagtgc tggcccagac
     1201 tatggtgccc tctagtgcta tggtgcctct ggcccagcca cctgctccag cccctgtgct
     1261 gaccccagga ccaccccagt cactgagcgc tccagtgccc aagtctacac aggccggcga
     1321 ggggactctg agtgaagctc tgctgcacct gcagttcgac gctgatgagg acctgggagc
     1381 tctgctgggg aacagcaccg atcccggagt gttcacagat ctggcctccg tggacaactc
     1441 tgagtttcag cagctgctga atcagggcgt gtccatgtct catagtacag ccgaaccaat
     1501 gctgatggag taccccgaag ccattacccg gctggtgacc ggcagccagc ggccccccga
     1561 ccccgctcca actcccctgg gaaccagcgg cctgcctaat gggctgtccg gagatgaaga
     1621 cttctcaagc atcgctgata tggactttag tgccctgctg tcacagattt cctctagtgg
     1681 gcagggagga ggtggaagcg gcttcagcgt ggacaccagt gccctgctgg acctgttcag
     1741 cccctcggtg accgtgcccg acatgagcct gcctgacctt gacagcagcc tggccagtat
     1801 ccaagagctc ctgtctcccc aggagccccc caggcctccc gaggcagaga acagcagccc
     1861 ggattcaggg aagcagctgg tgcactacac agcgcagccg ctgttcctgc tggaccccgg
     1921 ctccgtggac accgggagca acgacctgcc ggtgctgttt gagctgggag agggctccta
     1981 cttctccgaa ggggacggct tcgccgagga ccccaccatc tccctgctga caggctcgga
     2041 gcctcccaaa gccaaggacc ccactgtctc cgctagcggc atcgattgat taattaagaa
     2101 ttcgacccag ctttcttgta caaagtggtg aattcaagct tcggggacta gtcatatgat
     2161 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     2221 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     2281 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     2341 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     2401 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     2461 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     2521 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc
     2581 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     2641 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     2701 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca caattggaat
     2761 tcaataaaag atctttattt tcattagatc tgtgtgttgg ttttttgtgt gcggccgcag
     2821 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     2881 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     2941 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3001 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     3061 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     3121 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3181 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3241 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3301 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3361 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     3421 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     3481 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3541 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3601 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3661 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3721 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3781 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3841 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3901 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3961 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     4021 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     4081 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     4141 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     4201 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     4261 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4321 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4381 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4441 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4501 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4561 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     4621 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4681 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4741 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4801 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4861 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4921 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4981 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5041 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5101 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5161 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5221 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5281 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5341 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5401 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5461 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5521 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct
     5581 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     5641 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagaac
     5701 gcgtgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     5761 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     5821 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     5881 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     5941 aggacgaaac accggaagag cgagctcttc tgtttcagag ctaggccagc atgaggatca
     6001 cccatgcctg cagggcctag caagttgaaa taaggctagt ccgttatcaa cttggccagc
     6061 atgaggatca cccatgcctg cagggccaag tggcaccgag tcggtgcttt tttgcatatg
     6121 ctagtgcgcg ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     6181 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     6241 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     6301 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     6361 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     6421 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     6481 ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct
     6541 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg
     6601 ggggggggg
//
