LOCUS       Exported                5919 bp ds-DNA     circular SYN 07-AUG-2025
DEFINITION  AAV vector expressing Myc-tagged SpCas9 under the EFS promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5919)
  AUTHORS   Nishiyama J, Mikuni T, Yasuda R
  TITLE     Virus-Mediated Genome Editing via Homology-Directed Repair in 
            Mitotic and Postmitotic Cells in Mammalian Brain.
  JOURNAL   Neuron. 2017 Oct 18. pii: S0896-6273(17)30933-9. doi: 
            10.1016/j.neuron.2017.10.004.
  PUBMED    29056297
REFERENCE   2  (bases 1 to 5919)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 10, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
REFERENCE   3  (bases 1 to 5919)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Aug 7, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5919
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          join(3771..5919,1..787)
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      1..589
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of  
                     replication"
     repeat_region   651..780
                     /label=ITR
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_feature    join(788..815,1881..1992,2968..2991)
                     /label=phagemid pBluescriptII SK(+) [Split]
                     /note="phagemid pBluescriptII SK(+) [Split]"
     promoter        join(907..1449,1881..1984)
                     /label=GFA-ABC1D
                     /note="GFAP promoter of 681 bp from Dr M. Brenner 
                     corresponding to GFAP promoter sequences -1757 to -1256 / 
                     -132 to +47 [Split]"
     misc_feature    1485..1877
                     /label=(B)3
                     /note="/(B)3="
     misc_feature    2036..2063
                     /label=phagemid pBluescriptII SK(+) [Split]
                     /note="phagemid pBluescriptII SK(+) [Split]"
     misc_feature    2110..2941
     gene            2124..2231
                     /label=Mito
     CDS             2232..2909
                     /codon_start=1
                     /label=Timer
                     /note="Timer"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     gene            2544..2546
                     /label=Mutation V105A (T to C)
                     /label=Mutation V104A (T to C)
     gene            2820
                     /label=mutation S197T (T to A)
     misc_feature    2942..2967
     misc_feature    2942..2967
     misc_feature    2942..2959
     misc_feature    2942..2959
     misc_feature    2942..2959
     misc_feature    2942..2959
     misc_feature    2964..2967
     repeat_region   2968..3770
     repeat_region   2968..2991
     repeat_region   2968..2991
     gene            2992
                     /label=EGFP=
                     /note="EGFP="
     misc_feature    3624..3728
                     /label=miR124T(N)
                     /note="miR124T Naldini"
     polyA_signal    3776..3824
                     /label=synthetic polyadenylation signal
                     /note="synthetic polyadenylation signal"
     repeat_region   3832..3972
                     /label=ITR
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus  
                     serotype 2"
     repeat_region   3832..3961
                     /label=AAV2 ITR
     rep_origin      4047..4502
                     /label=f1 ori
                     /note="""f1 bacteriophage origin of replication"
                     /note="arrow  indicates direction of (+) strand 
                     synthesis"""
     primer_bind     complement(4519..4538)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable  
                     marker"
     promoter        4784..4888
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4889..5749
                     /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(5107..5126)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggcctcta gtggatcccc cgggctgcag gaattatcac aagtttgtac aaaaaagcag
      841 gctccaccat gggaaccaat tcagtcgact ggatccggta ccgaattcga gctcggtacc
      901 agatctaaca tatcctggtg tggagtaggg gacgctgctc tgacagaggc tcgggggcct
      961 gagctggctc tgtgagctgg ggaggaggca gacagccagg ccttgtctgc aagcagacct
     1021 ggcagcattg ggctggccgc cccccagggc ctcctcttca tgcccagtga atgactcacc
     1081 ttggcacaga cacaatgttc ggggtgggca cagtgcctgc ttcccgccgc accccagccc
     1141 ccctcaaatg ccttccgaga agcccattga gcagggggct tgcattgcac cccagcctga
     1201 cagcctggca tcttgggata aaagcagcac agccccctag gggctgccct tgctgtgtgg
     1261 cgccaccggc ggtggagaac aaggctctat tcagcctgtg cccaggaaag gggatcaggg
     1321 gatgcccagg catggacagt gggtggcagg gggggagagg agggctgtct gcttcccaga
     1381 agtccaagga cacaaatggg tgaggggaga gctctcccca tagctgggct gcggcccaac
     1441 cccaccccct caggctatgc cagggggtgt tgccaggggc acccgatctt cctcttcatg
     1501 cccagtgaat gactcacctt ggcacagaca caatgttcgg ggtgggcaca gtgcctgctt
     1561 cccgccgcac cccagccccc ctcaaatgcc ttccgagaag cccattgagc aggcccgatc
     1621 ttcctcttca tgcccagtga atgactcacc ttggcacaga cacaatgttc ggggtgggca
     1681 cagtgcctgc ttcccgccgc accccagccc ccctcaaatg ccttccgaga agcccattga
     1741 gcaggcccga tcttcctctt catgcccagt gaatgactca ccttggcaca gacacaatgt
     1801 tcggggtggg cacagtgcct gcttcccgcc gcaccccagc ccccctcaaa tgccttccga
     1861 gaagcccatt gagcaggccc gggcatcgcc agtctagccc actccttcat aaagccctcg
     1921 catcccagga gcgagcagag ccagagcagg ttggagagga gacgcatcac ctccgctgct
     1981 cgcggggatc ctctagagtc gacggatccg gggaattcgc ggccgcactc gagatctagt
     2041 ggatcccccg ggctgcagga attatcacaa gtttgtacaa aaaagcaggc tccgcggccg
     2101 cccccttcac cagatctgct agcatgtccg tcctgacgcc gctgctgctg cggggcttga
     2161 caggctcggc ccggcggctc ccagtgccgc gcgccaagat ccattcgttg ggggatccac
     2221 cggtcgccac catggcctcc tccgagaacg tcatcaccga gttcatgcgc ttcaaggtgc
     2281 gcatggaggg caccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     2341 cctacgaggg ccacaacacc gtgaagctga aggtgaccaa gggcggcccc ctgcccttcg
     2401 cctgggacat cctgtccccc cagttccagt acggctccaa ggtgtacgtg aagcaccccg
     2461 ccgacatccc cgactacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     2521 tgaacttcga ggacggcggc gtggcgaccg tgacccagga ctcctccctg caggacggct
     2581 gcttcatcta caaggtgaag ttcatcggcg tgaacttccc ctccgacggc cccgtgatgc
     2641 agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc
     2701 tgaagggcga gatccacaag gccctgaagc tgaaggacgg cggccactac ctggtggagt
     2761 tcaagtccat ctacatggcc aagaagcccg tgcagctgcc cggctactac tacgtggaca
     2821 ccaagctgga catcacctcc cacaacgagg actacaccat cgtggagcag tacgagcgca
     2881 ccgagggccg ccaccacctg ttcctgtagc tcgagaaggg tgggcgcgcc gacccagctt
     2941 tcttgtacaa agtggtgata attcgatatc aagcttatcg ataccgtcga cctcgaggga
     3001 attccgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3061 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3121 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3181 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3241 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3301 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3361 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat
     3421 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3481 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3541 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     3601 gggaattcga gctcggtacc taatggcatt caccgcgtgc cttaattcga atggcattca
     3661 ccgcgtgcct taaacgcgtt ggcattcacc gcgtgcctta aatgcattgg cattcaccgc
     3721 gtgccttaag gtacctttaa gaccaatgac ttacaaggca gctgtagatc aattcaataa
     3781 aagatcttta ttttcattag atctgtgtgt tggttttttg tgtgcggccg caggaacccc
     3841 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     3901 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     3961 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4021 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     4081 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     4141 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4201 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4261 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4321 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     4381 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     4441 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     4501 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     4561 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4621 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4681 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4741 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4801 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4861 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4921 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4981 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5041 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5101 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5161 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5221 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5281 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5341 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5401 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5461 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5521 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5581 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     5641 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5701 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5761 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5821 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5881 ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
