LOCUS       sequence-430319-        6954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV plasmid containing sgCTG (target sequence: (CUG)6) driven by a 
            U6 promoter and eGFP under the control of the CMV promoter. Used for
            in vivo experiments..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6954)
  AUTHORS   Alvaro Murillo, Melanie Alpaugh, Meghan Larin, Emma L. Randall, 
            Laura Heraty, Ruban Rex Durairaj, Alys N. Aston, Alysha S. Taylor, 
            Alex Mas Monteys, Nina Stoberl, Aeverie E. R. Heuchan, Pascale 
            Aeschlimann, Soumyasree Bhattacharyya, Nicholas D. Allen, Jack 
            Puymirat, Beverly L. Davidson, Francesca Cicchetti, Mariah Lelos, 
            Vincent Dion
  TITLE     Cas9 nickase-mediated contraction of CAG/CTG repeats at multiple 
            disease loci
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 6954)
  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: "bioRxiv"
COMMENT     Sequence Label: pAAV-U6-sgCTG-CMV-GFP
FEATURES             Location/Qualifiers
     source          1..6954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     mobile_element  183..348
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     polyA_signal    377..511
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   678..818
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   689..818
                     /label=AAV2 ITR
     misc_RNA        complement(976..1051)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1072..1320)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1130..1149)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1300..1320)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     enhancer        1398..1701
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1702..1905
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1856..1876
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1934..1943
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1940..1942,1943..1945,1946..2659)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1940..1942,1943..1945,1946..2656)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1985..2006)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2246..2265)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2593..2614
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2673..2692)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2674..2692)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2718..2852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2934..3063
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        join(3403..3408,3409..3425,3426..3431)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3620..4153
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     mobile_element  4220..4444
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     rep_origin      complement(4749..5337)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4829..4848)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5508..6299,6300..6368))
                     /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     6131..6150
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6369..6473)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6499..6953)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6636..6657)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6847..6866
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccattcgcca ttcaggctgc aaataagcgt tgatattcag tcaattacaa acattaataa
       61 cgaagagatg acagaaaaat tttcattctg tgacagagaa aaagtagccg aagatgacgg
      121 tttgtcacat ggagttggca ggatgtttga ttaaaaacat aacaggaaga aaaatgcccc
      181 gctgtgggcg gacaaaatag ttgggaactg ggaggggtgg aaatggagtt tttaaggatt
      241 atttagggaa gagtgacaaa atagatggga actgggtgta gcgtcgtaag ctaatacgaa
      301 aattaaaaat gacaaaatag tttggaacta gatttcactt atctggttcg gatctcctag
      361 gcgatatcag tgatcagatc cagacatgat aagatacatt gatgagtttg gacaaaccac
      421 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
      481 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
      541 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
      601 tatggctgat tatgatcctc tagtacttct cgacaagctt acattattga agcatttatc
      661 agggttattg tctcagacct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg
      721 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
      781 cagagaggga gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc
      841 atgctactta tctacgtagc catgctctag agcggccgca cgcgtactag agccatttgt
      901 ctgcagaatt ggcgcacgcg ctaaaaacgg actagcctta ttttaacttg ctatttctag
      961 ctctaaaaca aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct
     1021 tattttaact tgctatttct agctctaaaa cgcagcagca gcagcagcag cggtgtttcg
     1081 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
     1141 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
     1201 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat
     1261 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc
     1321 acatgaaacg aattctcgac ttattaatag taatcaatta cggggtcatt agttcatagc
     1381 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     1441 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     1501 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     1561 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     1621 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     1681 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     1741 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     1801 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     1861 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
     1921 cagatccgct agcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1981 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2041 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2101 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2161 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2221 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2281 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2341 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2401 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2461 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2521 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     2581 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     2641 ggacgagctg tacaagtaag gtacccaatt cgccctatag tgagtcgtat tacgcgcgca
     2701 gcggccgacc atggcccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2761 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     2821 aactcatcaa tgtatcttat catgtctgga tctccggaca cgtgcggacc gagcggccgc
     2881 tctagagcat ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc
     2941 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     3001 accaaaggtc gcccgacgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     3061 aggcatgcaa gctgtagcca accactagaa ctatagctag agtcctgggc gaacaaacga
     3121 tgctcgcctt ccagaaaacc gaggatgcga accacttcat ccggggtcag caccaccggc
     3181 aagcgccgcg acggccgagg tcttccgatc tcctgaagcc agggcagatc cgtgcacagc
     3241 accttgccgt agaagaacag caaggccgcc aatgcctgac gatgcgtgga gaccgaaacc
     3301 ttgcgctcgt tcgccagcca ggacagaaat gcctcgactt cgctgctgcc caaggttgcc
     3361 gggtgacgca caccgtggaa acggatgaag gcacgaaccc agttgacata agcctgttcg
     3421 gttcgtaaac tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc
     3481 gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt
     3541 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
     3601 atgttatgga gcagcaacga tgttacgcag cagcaacgat gttacgcagc agggcagtcg
     3661 ccctaaaaca aagttaggtg gctcaagtat gggcatcatt cgcacatgta ggctcggccc
     3721 tgaccaagtc aaatccatgc gggctgctct tgatcttttc ggtcgtgagt tcggagacgt
     3781 agccacctac tcccaacatc agccggactc cgattacctc gggaacttgc tccgtagtaa
     3841 gacattcatc gcgcttgctg ccttcgacca agaagcggtt gttggcgctc tcgcggctta
     3901 cgttctgccc aagtttgagc agccgcgtag tgagatctat atctatgatc tcgcagtctc
     3961 cggcgagcac cggaggcagg gcattgccac cgcgctcatc aatctcctca agcatgaggc
     4021 caacgcgctt ggtgcttatg tgatctacgt gcaagcagat tacggtgacg atcccgcagt
     4081 ggctctctat acaaagttgg gcatacggga agaagtgatg cactttgata tcgacccaag
     4141 taccgccacc taacaattcg ttcaagccga gatcggcttc ccggccgcgg agttgttcgg
     4201 taaattgtca caacgccgcg aatatagtct ttaccatgcc cttggccacg cccctcttta
     4261 atacgacggg caatttgcac ttcagaaaat gaagagtttg ctttagccat aacaaaagtc
     4321 cagtatgctt tttcacagca taactggact gatttcagtt tacaactatt ctgtctagtt
     4381 taagacttta ttgtcatagt ttagatctat tttgttcagt ttaagacttt attgtccgcc
     4441 cacacccgct tacgcagggc atccatttat tactcaaccg taaccgattt tgccaggtta
     4501 cgcggctggt ctatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     4561 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4621 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4681 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4741 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4801 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4861 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4921 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4981 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5041 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5101 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5161 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5221 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5281 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5341 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5401 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5461 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5521 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5581 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5641 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5701 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5761 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5821 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5881 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5941 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6001 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6061 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6121 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6181 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6241 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6301 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6361 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6421 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6481 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     6541 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     6601 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     6661 taagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     6721 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     6781 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     6841 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     6901 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtc
//
