LOCUS       sequence-378841-        4758 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-KamiCas9 vector expressing expressing thew GFP reporter gene and
            sgHTT and sgCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4758)
  AUTHORS   Duarte F, Ramosaj M, Hasanovic E, Regio S, Sipion M, Rey M, Deglon N
  TITLE     Semi-automated workflows to quantify AAV transduction in various 
            brain areas and predict gene editing outcome for neurological 
            disorders.
  JOURNAL   Mol Ther Methods Clin Dev. 2023 Mar 27;29:254-270. doi: 
            10.1016/j.omtm.2023.03.013. eCollection 2023 Jun 8.
  PUBMED    37090478
REFERENCE   2  (bases 1 to 4758)
  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"; journalName: "Mol Ther 
            Methods Clin Dev."; date: "2023-03-27"; volume: "29:254-270. doi"; 
            pages: " 10.1016/j.omtm.2023.03.013. eCollection 2023 Jun 8"
COMMENT     Sequence Label: pAAV2ss-EFS-GFP-synPolyA-U6-sgHTT1-U6-sgCas9
FEATURES             Location/Qualifiers
     source          1..4758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   14..143
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        204..415
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      424..433
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(430..432,433..435,436..1146)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(430..432,433..435,436..1146)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(475..496)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(736..755)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1083..1104
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1209..1257
                     /note="synthetic polyadenylation signal"
     promoter        1273..1521
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        1653..1901
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   2034..2174
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2034..2163
                     /label=AAV2 ITR
     rep_origin      2249..2704
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2336..2355)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2546..2567
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2721..2740)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2840..2862
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2900..2918)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2986..3090
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3091..3159,3160..3951)
                     /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(3309..3328)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4122..4710
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4611..4630
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg
       61 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc
      121 caactccatc actaggggtt cctgcggcct ctagaaagct taggtcttga aaggagtggg
      181 aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
      241 ggggggaggg gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga
      301 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
      361 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggaccg
      421 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      481 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
      541 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
      601 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
      661 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
      721 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
      781 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
      841 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
      901 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
      961 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1021 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1081 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1141 tacaagtccg gactcagatc tcgactagct agtagctagc tagctagtcg agctcaagct
     1201 tcgaattcaa taaaagatct ttattttcat tagatctgtg tgttggtttt ttgtgtgcgg
     1261 ccgctgggat ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag
     1321 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     1381 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     1441 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     1501 ttgtggaaag gacgaaacac cgaccctgga aaagctgatg agtttcagag ctatgctgga
     1561 aacagcatag caagttgaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     1621 tcggtgcttt ttttggatcc aagcttccat gggagggcct atttcccatg attccttcat
     1681 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     1741 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1801 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     1861 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgtcgccgaa gaaaaagcgc
     1921 agtttcagag ctatgctgga aacagcatag caagttgaaa taaggctagt ccgttatcaa
     1981 cttgaaaaag tggcaccgag tcggtgcttt ttttcatagc catgggcggc cgcaggaacc
     2041 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     2101 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     2161 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2221 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     2281 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     2341 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2401 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2461 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2521 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2581 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     2641 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     2701 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     2761 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2821 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     2881 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     2941 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3001 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3061 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3121 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3181 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3241 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3301 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3361 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3421 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3481 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3541 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3601 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3661 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3721 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3781 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     3841 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3901 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3961 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4021 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4081 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4141 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4201 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4261 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4321 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4381 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4441 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4501 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4561 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4621 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4681 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4741 ctggcctttt gctggcct
//
