LOCUS       Exported                6120 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 6120)
  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 6120)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 10, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
REFERENCE   3  (bases 1 to 6120)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Aug 7, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6120
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          join(3972..6120,1..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"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=ITR
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_feature    788..815
                     /label=phagemid pBluescriptII SK(+) [Split]
                     /note="phagemid pBluescriptII SK(+) [Split]"
     misc_feature    888..1974
     promoter        join(889..1431,1863..1966)
                     /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    1467..1859
                     /label=(B)3
                     /note="/(B)3="
     misc_feature    1863..1974
     gene            1980..3061
                     /label=/nls-CRE=
                     /note="/nls-CRE="
     CDS             2377..2917
                     /codon_start=1
                     /label=ORF2, put. cre protein (aa 1-343) [Split]
                     /note="ORF2, put. cre protein (aa 1-343) [Split]"
                     /translation="EKKTLMPVNVQNRL*RSNALISTRFVHSWKIAIAARIYVIWHFWG
                     LLITPCYV*PKLPGSGLKISHVLTVGEC*SILAERKRWLAPQV*RRHLAWG*LNWSSDG
                     FPSLV*LMIRITTCFAGSEKMVLPRHLPPASYQLAPWKGFLKQLID*FTALRMTLVRDT
                     WPGLDTVPVSEPREIWP"
     repeat_region   3143..3971
     repeat_region   3143..3192
     repeat_region   3143..3192
     repeat_region   3143..3192
                     /label=RFA
     misc_feature    3143..3160
     misc_feature    3165..3192
     gene            3193
                     /label=EGFP=
                     /note="EGFP="
     misc_feature    3825..3929
                     /label=miR124T(N)
                     /note="miR124T Naldini"
     polyA_signal    3977..4025
                     /label=synthetic polyadenylation signal
                     /note="synthetic polyadenylation signal"
     repeat_region   4033..4173
                     /label=ITR
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus  
                     serotype 2"
     repeat_region   4033..4162
                     /label=AAV2 ITR
     rep_origin      4248..4703
                     /label=f1 ori
                     /note="""f1 bacteriophage origin of replication"
                     /note="arrow  indicates direction of (+) strand 
                     synthesis"""
     primer_bind     complement(4335..4354)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4545..4566
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4720..4739)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable  
                     marker"
     primer_bind     4839..4861
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4899..4917)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward  
                     primer"
     promoter        4985..5089
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5090..5950
                     /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(5308..5327)
                     /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 ccagatctaa catatcctgg
      901 tgtggagtag gggacgctgc tctgacagag gctcgggggc ctgagctggc tctgtgagct
      961 ggggaggagg cagacagcca ggccttgtct gcaagcagac ctggcagcat tgggctggcc
     1021 gccccccagg gcctcctctt catgcccagt gaatgactca ccttggcaca gacacaatgt
     1081 tcggggtggg cacagtgcct gcttcccgcc gcaccccagc ccccctcaaa tgccttccga
     1141 gaagcccatt gagcaggggg cttgcattgc accccagcct gacagcctgg catcttggga
     1201 taaaagcagc acagccccct aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga
     1261 acaaggctct attcagcctg tgcccaggaa aggggatcag gggatgccca ggcatggaca
     1321 gtgggtggca gggggggaga ggagggctgt ctgcttccca gaagtccaag gacacaaatg
     1381 ggtgagggga gagctctccc catagctggg ctgcggccca accccacccc ctcaggctat
     1441 gccagggggt gttgccaggg gcacccgatc ttcctcttca tgcccagtga atgactcacc
     1501 ttggcacaga cacaatgttc ggggtgggca cagtgcctgc ttcccgccgc accccagccc
     1561 ccctcaaatg ccttccgaga agcccattga gcaggcccga tcttcctctt catgcccagt
     1621 gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct gcttcccgcc
     1681 gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggccc gatcttcctc
     1741 ttcatgccca gtgaatgact caccttggca cagacacaat gttcggggtg ggcacagtgc
     1801 ctgcttcccg ccgcacccca gcccccctca aatgccttcc gagaagccca ttgagcaggc
     1861 ccgggcatcg ccagtctagc ccactccttc ataaagccct cgcatcccag gagcgagcag
     1921 agccagagca ggttggagag gagacgcatc acctccgctg ctcgcgggga tcctctagtg
     1981 gatctcccgg gaccatgccc aagaagaaga ggaaggtgtc caatttactg accgtacacc
     2041 aaaatttgcc tgcattaccg gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg
     2101 acatgttcag ggatcgccag gcgttttctg agcatacctg gaaaatgctt cagtccgttt
     2161 gccggtcgtg ggcggcatgg tgcaagttga ataaccggaa atggtttccc gcagaacctg
     2221 aagatgttcg cgattatctt ctatatcttc aggcgcgcgg tctggcagta aaaactatcc
     2281 agcaacattt gggccagcta aacatgcttc atcgtcggtc cgggctgcca cgaccaagtg
     2341 acagcaatgc tgtttcactg gttatgcggc ggatccgaaa agaaaacgtt gatgccggtg
     2401 aacgtgcaaa acaggctcta gcgttcgaac gcactgattt cgaccaggtt cgttcactca
     2461 tggaaaatag cgatcgctgc caggatatac gtaatctggc atttctgggg attgcttata
     2521 acaccctgtt acgtatagcc gaaattgcca ggatcagggt taaagatatc tcacgtactg
     2581 acggtgggag aatgttaatc catattggca gaacgaaaac gctggttagc accgcaggtg
     2641 tagagaaggc acttagcctg ggggtaacta aactggtcga gcgatggatt tccgtctctg
     2701 gtgtagctga tgatccgaat aactacctgt tttgccgggt cagaaaaaat ggtgttgccg
     2761 cgccatctgc caccagccag ctatcaactc gcgccctgga agggattttt gaagcaactc
     2821 atcgattgat ttacggcgct aaggatgact ctggtcagag atacctggcc tggtctggac
     2881 acagtgcccg tgtcggagcc gcgcgagata tggcccgcgc tggagtttca ataccggaga
     2941 tcatgcaagc tggtggctgg accaatgtaa atattgtcat gaactatatc cgtaacctgg
     3001 atagtgaaac aggggcaatg gtgcgcctgc tggaagatgg cgattagcca ttaacgcgta
     3061 aatgattgct ataattattt gatatttatg gtgacatacg agggaattcg cggccgcact
     3121 cgagatatct agacccagct ttcttgtaca aagtggtgat aattcgatat caagcttatc
     3181 gataccgtcg acctcgaggg aattccgata atcaacctct ggattacaaa atttgtgaaa
     3241 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3301 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     3361 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     3421 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     3481 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     3541 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     3601 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     3661 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     3721 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     3781 tttgggccgc ctccccgcat cgggaattcg agctcggtac ctaatggcat tcaccgcgtg
     3841 ccttaattcg aatggcattc accgcgtgcc ttaaacgcgt tggcattcac cgcgtgcctt
     3901 aaatgcattg gcattcaccg cgtgccttaa ggtaccttta agaccaatga cttacaaggc
     3961 agctgtagat caattcaata aaagatcttt attttcatta gatctgtgtg ttggtttttt
     4021 gtgtgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     4081 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4141 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     4201 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     4261 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4321 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4381 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4441 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     4501 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4561 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     4621 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4681 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     4741 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4801 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4861 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     4921 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     4981 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5041 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5101 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5161 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5221 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5281 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5341 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5401 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5461 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     5521 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     5581 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     5641 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     5701 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5761 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
     5821 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5881 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5941 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6001 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6061 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
