LOCUS       sequence-148033-        4787 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  A single vector AAV-Cas9 system containing SaCas9 under EFs 
            promoter, gRNAs targeting Dmd introns 22 and 23.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4787)
  AUTHORS   Tabebordbar M, Zhu K, Cheng JK, Chew WL, Widrick JJ, Yan WX, Maesner
            C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church 
            GM, Wagers AJ
  TITLE     In vivo gene editing in dystrophic mouse muscle and muscle stem 
            cells.
  JOURNAL   Science. 2016 Jan 22;351(6271):407-11. doi: 10.1126/science.aad5177.
            Epub 2015 Dec 31.
  PUBMED    26721686
REFERENCE   2  (bases 1 to 4787)
  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: 
            "10.1126/science.aad5177"; journalName: "Science"; date: 
            "2016-01-22- 22"; volume: "351"; issue: "6271"; pages: "407-11"
COMMENT     Sequence Label: pZac2.1 EFs-SaCas9-U6-Sa DMDR7-U6-SaDMDL2
FEATURES             Location/Qualifiers
     source          1..4787
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     repeat_region   21..150
                     /label=AAV2 ITR
     promoter        226..437
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             462..3620
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE
                     GRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEE
                     FSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGE
                     VRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFG
                     WKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKF
                     QIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKE
                     IIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKA
                     INLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSI
                     KVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENA
                     KYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVK
                     QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRF
                     SVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER
                     NKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEI
                     FITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKD
                     NDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKK
                     DNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKN
                     LDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLL
                     NRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQ
                     IIKKG"
     CDS             3624..3644
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3648..3668
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3672..3692
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3699..3725
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3735..3856
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3772..3791)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3826..3845
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3863..4111
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        4232..4480
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   4651..4780
                     /label=AAV2 ITR
ORIGIN
        1 taattaaggc cttaattagg ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc
       61 ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg
      121 gagtggccaa ctccatcact aggggttcct tgtagttaat gattaacccg ccatgctact
      181 tatctacgaa ttcgagctcg gtacccggct ccggtgcccg tcagtgggca gagcgcacat
      241 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa
      301 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
      361 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
      421 ttgccgccag aacacaggac cggttctaga gcgctgccac catgaaaagg aactacattc
      481 tggggctgga catcgggatt acaagcgtgg ggtatgggat tattgactat gaaacaaggg
      541 acgtgatcga cgcaggcgtc agactgttca aggaggccaa cgtggaaaac aatgagggac
      601 ggagaagcaa gaggggagcc aggcgcctga aacgacggag aaggcacaga atccagaggg
      661 tgaagaaact gctgttcgat tacaacctgc tgaccgacca ttctgagctg agtggaatta
      721 atccttatga agccagggtg aaaggcctga gtcagaagct gtcagaggaa gagttttccg
      781 cagctctgct gcacctggct aagcgccgag gagtgcataa cgtcaatgag gtggaagagg
      841 acaccggcaa cgagctgtct acaaaggaac agatctcacg caatagcaaa gctctggaag
      901 agaagtatgt cgcagagctg cagctggaac ggctgaagaa agatggcgag gtgagagggt
      961 caattaatag gttcaagaca agcgactacg tcaaagaagc caagcagctg ctgaaagtgc
     1021 agaaggctta ccaccagctg gatcagagct tcatcgatac ttatatcgac ctgctggaga
     1081 ctcggagaac ctactatgag ggaccaggag aagggagccc cttcggatgg aaagacatca
     1141 aggaatggta cgagatgctg atgggacatt gcacctattt tccagaagag ctgagaagcg
     1201 tcaagtatgc ttataacgca gatctgtaca acgccctgaa tgacctgaac aacctggtca
     1261 tcaccaggga tgaaaacgag aaactggaat actatgagaa gttccagatc atcgaaaacg
     1321 tgtttaagca gaagaaaaag cctacactga aacagattgc taaggagatc ctggtcaacg
     1381 aagaggacat caagggctac cgggtgacaa gcactggaaa accagagttc accaatctga
     1441 aagtgtatca cgatattaag gacatcacag cacggaaaga aatcattgag aacgccgaac
     1501 tgctggatca gattgctaag atcctgacta tctaccagag ctccgaggac atccaggaag
     1561 agctgactaa cctgaacagc gagctgaccc aggaagagat cgaacagatt agtaatctga
     1621 aggggtacac cggaacacac aacctgtccc tgaaagctat caatctgatt ctggatgagc
     1681 tgtggcatac aaacgacaat cagattgcaa tctttaaccg gctgaagctg gtcccaaaaa
     1741 aggtggacct gagtcagcag aaagagatcc caaccacact ggtggacgat ttcattctgt
     1801 cacccgtggt caagcggagc ttcatccaga gcatcaaagt gatcaacgcc atcatcaaga
     1861 agtacggcct gcccaatgat atcattatcg agctggctag ggagaagaac agcaaggacg
     1921 cacagaagat gatcaatgag atgcagaaac gaaaccggca gaccaatgaa cgcattgaag
     1981 agattatccg aactaccggg aaagagaacg caaagtacct gattgaaaaa atcaagctgc
     2041 acgatatgca ggagggaaag tgtctgtatt ctctggaggc catccccctg gaggacctgc
     2101 tgaacaatcc attcaactac gaggtcgatc atattatccc cagaagcgtg tccttcgaca
     2161 attcctttaa caacaaggtg ctggtcaagc aggaagagaa ctctaaaaag ggcaatagga
     2221 ctcctttcca gtacctgtct agttcagatt ccaagatctc ttacgaaacc tttaaaaagc
     2281 acattctgaa tctggccaaa ggaaagggcc gcatcagcaa gaccaaaaag gagtacctgc
     2341 tggaagagcg ggacatcaac agattctccg tccagaagga ttttattaac cggaatctgg
     2401 tggacacaag atacgctact cgcggcctga tgaatctgct gcgatcctat ttccgggtga
     2461 acaatctgga tgtgaaagtc aagtccatca acggcgggtt cacatctttt ctgaggcgca
     2521 aatggaagtt taaaaaggag cgcaacaaag ggtacaagca ccatgccgaa gatgctctga
     2581 ttatcgcaaa tgccgacttc atctttaagg agtggaaaaa gctggacaaa gccaagaaag
     2641 tgatggagaa ccagatgttc gaagagaagc aggccgaatc tatgcccgaa atcgagacag
     2701 aacaggagta caaggagatt ttcatcactc ctcaccagat caagcatatc aaggatttca
     2761 aggactacaa gtactctcac cgggtggata aaaagcccaa cagagagctg atcaatgaca
     2821 ccctgtatag tacaagaaaa gacgataagg ggaataccct gattgtgaac aatctgaacg
     2881 gactgtacga caaagataat gacaagctga aaaagctgat caacaaaagt cccgagaagc
     2941 tgctgatgta ccaccatgat cctcagacat atcagaaact gaagctgatt atggagcagt
     3001 acggcgacga gaagaaccca ctgtataagt actatgaaga gactgggaac tacctgacca
     3061 agtatagcaa aaaggataat ggccccgtga tcaagaagat caagtactat gggaacaagc
     3121 tgaatgccca tctggacatc acagacgatt accctaacag tcgcaacaag gtggtcaagc
     3181 tgtcactgaa gccatacaga ttcgatgtct atctggacaa cggcgtgtat aaatttgtga
     3241 ctgtcaagaa tctggatgtc atcaaaaagg agaactacta tgaagtgaat agcaagtgct
     3301 acgaagaggc taaaaagctg aaaaagatta gcaaccaggc agagttcatc gcctcctttt
     3361 acaacaacga cctgattaag atcaatggcg aactgtatag ggtcatcggg gtgaacaatg
     3421 atctgctgaa ccgcattgaa gtgaatatga ttgacatcac ttaccgagag tatctggaaa
     3481 acatgaatga taagcgcccc cctcgaatta tcaaaacaat tgcctctaag actcagagta
     3541 tcaaaaagta ctcaaccgac attctgggaa acctgtatga ggtgaagagc aaaaagcacc
     3601 ctcagattat caaaaagggc gaccccaaga agaagaggaa ggttgatcca aaaaagaaga
     3661 gaaaggtaga tcccaagaaa aaaagaaaag tcggatccta cccatacgat gttccagatt
     3721 acgcttaaga attcaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3781 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3841 tcatcaatgt atcttaggta ccgagggcct atttcccatg attccttcat atttgcatat
     3901 acgatacaag gctgttagag agataattag aattaatttg actgtaaaca caaagatatt
     3961 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     4021 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     4081 tttatatatc ttgtggaaag gacgaaacac cgcagtaatg tgtcatacct tcgtttaagt
     4141 actctgtgct ggaaacagca cagaatctac ttaaacaagg caaaatgccg tgtttatctc
     4201 gtcaacttgt tggcgagatt tttttccgcg ggagggccta tttcccatga ttccttcata
     4261 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     4321 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     4381 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     4441 tttcttggct ttatatatct tgtggaaagg acgaaacacc gatataatag aaattattca
     4501 tgtttaagta ctctgtgctg gaaacagcac agaatctact taaacaaggc aaaatgccgt
     4561 gtttatctcg tcaacttgtt ggcgagattt ttttggggat cctctagagt cgagtagata
     4621 agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc
     4681 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4741 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ccttaat
//
