LOCUS       sequence-385198-        7348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal AAV genome plasmid encoding ABE8e + sgRNA to correct the 
            R403Q mutation in mice.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7348)
  AUTHORS   Reichart D, Newby GA, Wakimoto H, Lun M, Gorham JM, Curran JJ, 
            Raguram A, DeLaughter DM, Conner DA, Marsiglia JDC, Kohli S, Chmatal
            L, Page DC, Zabaleta N, Vandenberghe L, Liu DR, Seidman JG, Seidman 
            C
  TITLE     Efficient in vivo genome editing prevents hypertrophic 
            cardiomyopathy in mice.
  JOURNAL   Nat Med. 2023 Feb;29(2):412-421. doi: 10.1038/s41591-022-02190-7. 
            Epub 2023 Feb 16.
  PUBMED    36797483
REFERENCE   2  (bases 1 to 7348)
  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: 
            "10.1038/s41591-022-02190-7"; journalName: "Nat Med"; date: 
            "2023-02"; volume: "29"; issue: "2"; pages: "412-421"
COMMENT     Sequence Label: pABE8e-NG-403Q-Cterm-AAV
FEATURES             Location/Qualifiers
     source          1..7348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(125..142)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(125..141)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(134..156)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      283..738
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(370..389)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     580..601
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        764..868
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(869..937,938..1729)
                     /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(1087..1106)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1900..2488
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2389..2408
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2776..2797
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2812..2817,2818..2835,2836..2842)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2850..2866
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2855..2877
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2874..2890
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2874..2890
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2917..3046
                     /label=AAV2 ITR
     intron          3617..3749
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3732..3751
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        3794..3812
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3884..3904
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4010..6394
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(C)
                     /translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
                     IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
                     TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
                     ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
                     LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
                     KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
                     AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
                     QVNIVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKY
                     FDTTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             6437..6457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6522..6542)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    6718..6942
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(6956..7031)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(7053..7301)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7111..7130)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7281..7301)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   join(7325..7348,1..106)
                     /label=AAV2 ITR
ORIGIN
        1 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
       61 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta
      121 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
      181 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
      241 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
      301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
      541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
      601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
      661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
      721 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      781 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      841 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      901 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      961 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1021 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1081 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1141 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1201 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1261 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1321 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1381 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1441 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1501 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1561 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1621 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1681 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1741 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1801 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1861 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1921 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1981 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2041 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2101 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2161 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2221 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2281 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2341 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2401 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2461 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2521 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2581 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2641 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     2701 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2761 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     2821 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     2881 cagctatgac catgattacg ccagatttaa ttaaggctgc gcgctcgctc gctcactgag
     2941 gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     3001 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagacg
     3061 ggccccccct cgaggtcggg ataaaagcag tctgggcttt cacatgacag catctggggc
     3121 tgcggcagag ggtcgggtcc gaagcgctgc cttatcagcg tccccagccc tgggaggtga
     3181 cagctggctg gcttgtgtca gcccctcggg cactcacgta tctccgtccg acgggtttaa
     3241 aatagcaaaa ctctgaggcc acacaatagc ttgggcttat atgggctcct gtgggggaag
     3301 ggggagcacg gagggggccg gggccgctgc tgccaaaata gcagctcaca agtgttgcat
     3361 tcctctctgg gcgccgggca cattcctgct ggctctgccc gccccggggt gggcgccggg
     3421 gggaccttaa agcctctgcc ccccaaggag cccttcccag acagccgccg gcacccaccg
     3481 ctccgtggga cgatccccga agctctagag ctttattgcg gtagtttatc acagttaaat
     3541 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
     3601 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
     3661 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
     3721 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     3781 aggctagagt acttaatacg actcactata ggctagcctc gagaattcac gcgggaccgg
     3841 tgccaccatg aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa
     3901 agtcatcaag attgctacac ggaaatacct gggaaagcag aacgtgtacg acatcggcgt
     3961 ggagcgggat cacaacttcg ccctgaagaa tggctttatc gccagcaatt gcttcgactc
     4021 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     4081 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     4141 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     4201 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     4261 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     4321 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     4381 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     4441 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     4501 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     4561 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     4621 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     4681 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     4741 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     4801 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     4861 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     4921 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     4981 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     5041 taagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     5101 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     5161 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     5221 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     5281 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     5341 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     5401 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     5461 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     5521 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
     5581 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc cggcccaaga ggaacagcga
     5641 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgtgagccc
     5701 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
     5761 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
     5821 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
     5881 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
     5941 ccggttcctg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
     6001 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
     6061 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
     6121 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
     6181 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
     6241 tctgggagcc cctcgggcct tcaagtactt tgacaccacc atcgaccgga aggtgtaccg
     6301 gagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
     6361 gacacggatc gacctgtctc agctgggagg tgactctggc ggctcaaaaa gaaccgccga
     6421 cggcagcgaa ttcgagccca agaagaagag gaaagtctaa gatctgataa tcaacctctg
     6481 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     6541 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     6601 ttctcctcct tgtataaatc ctggttagtt cttgccacgg cggaactcat cgccgcctgc
     6661 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgcgactg
     6721 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     6781 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     6841 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     6901 aagacaatag caggcatgct ggggatgcgg tgggctctat ggctcgagaa aaaaagcacc
     6961 gactcggtgc cactttttca agttgataac ggactagcct tattttaact tgctatttct
     7021 agctctaaaa cttccccacc ttcacctgag gcggtgtttc gtcctttcca caagatatat
     7081 aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa
     7141 cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt
     7201 actaatatct ttgtgtttac agtcaaatta attctaatta tctctctaac agccttgtat
     7261 cgtatatgca aatatgaagg aatcatggga aataggccct cttcctgccc gaccttgcgg
     7321 ccgcaggaac ccctagtgat ggagttgg
//
