LOCUS       sequence-455681-        7991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encoding Nme2ABE8e and sgRNA cassette on a single AAV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7991)
  AUTHORS   Liu Z, Yang L, Yang Y, Li J, Chen Z, Guo C, Guo Q, Li Q, Zhao D, Hu 
            X, Gao F, Guo Y
  TITLE     ABE-Mediated Cardiac Gene Silencing via Single AAVs Requires DNA 
            Accessibility.
  JOURNAL   Circ Res. 2025 Jan 16. doi: 10.1161/CIRCRESAHA.124.325611.
  PUBMED    39817340
REFERENCE   2  (bases 1 to 7991)
  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: "Circ Res. 
            2025 Jan 16. doi: 10.1161/CIRCRESAHA.124.325611."
COMMENT     Sequence Label: pAAV-EFS-TadA8e-Nme2-U6-sgRNA scaffold
FEATURES             Location/Qualifiers
     source          1..7991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     primer_bind     complement(149..166)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(149..165)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(158..180)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      307..762
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(394..413)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     604..625
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        788..892
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(893..961,962..1753)
                     /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(1111..1130)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1924..2512
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2413..2432
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2800..2821
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2836..2841,2842..2859,2860..2866)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2874..2890
                     /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     2879..2901
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2898..2914
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2898..2914
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2941..3070
                     /label=AAV2 ITR
     promoter        3136..3347
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3397..3417
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7294..7314
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    7328..7552
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(7578..7670)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(7720..7968)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7778..7797)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7948..7968)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
       61 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
      121 gagcgcgcag ccttaattaa cctaattcac tggccgtcgt tttacaacgt cgtgactggg
      181 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
      241 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
      301 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
      361 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      421 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      481 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      541 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      601 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      661 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
      721 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
      781 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
      841 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
      901 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
      961 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1021 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1081 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1141 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1201 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1261 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1321 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1381 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     1441 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1501 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1561 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     1621 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     1681 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1741 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1801 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1861 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1921 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1981 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2041 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2101 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2161 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2221 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2281 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2341 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2401 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2461 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2521 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     2581 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2641 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     2701 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     2761 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     2821 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     2881 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccagat ttaattaagg
     2941 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
     3001 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
     3061 aggggttcct gcggcctcta gaattcgcta gctaggtctt gaaaggagtg ggaattggct
     3121 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     3181 gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     3241 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     3301 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggac cggtgccacc
     3361 atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtctct
     3421 gaggtggagt tttcccacga gtactggatg agacatgccc tgaccctggc caagagggca
     3481 cgggatgaga gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag agtgatcggc
     3541 gagggctgga acagagccat cggcctgcac gacccaacag cccatgccga aattatggcc
     3601 ctgagacagg gcggcctggt catgcagaac tacagactga ttgacgccac cctgtacgtg
     3661 acattcgagc cttgcgtgat gtgcgccggc gccatgatcc actctaggat cggccgcgtg
     3721 gtgtttggcg tgaggaactc aaaaagaggc gccgcaggct ccctgatgaa cgtgctgaac
     3781 taccccggca tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc
     3841 gccctgctgt gcgatttcta tcggatgcct agacaggtgt tcaatgctca gaagaaggcc
     3901 cagagctcca tcaactctgg aggatctagc ggaggatcct ctggcagcga gacaccagga
     3961 acaagcgagt cagcaacacc agagagcagt ggcggcagca gcggcggcag cgcagcattc
     4021 aagcctaacc caatcaatta catcctggga ctggcaatcg gaatcgcatc cgtgggatgg
     4081 gctatggtgg agatcgacga ggaggagaat cctatccggc tgatcgatct gggcgtgaga
     4141 gtgtttgaga gggccgaggt gccaaagacc ggcgattctc tggctatggc ccggagactg
     4201 gcacggagcg tgaggcgcct gacacggaga agggcacaca ggctgctgag ggcacgccgg
     4261 ctgctgaaga gagagggcgt gctgcaggca gcagacttcg atgagaatgg cctgatcaag
     4321 agcctgccaa acaccccctg gcagctgaga gcagccgccc tggacaggaa gctgacacca
     4381 ctggagtggt ctgccgtgct gctgcacctg atcaagcacc gcggctacct gagccagcgg
     4441 aagaacgagg gagagacagc agacaaggag ctgggcgccc tgctgaaggg agtggccaac
     4501 aatgcccacg ccctgcagac cggcgatttc aggacacctg ccgagctggc cctgaataag
     4561 tttgagaagg agtccggcca catcagaaac cagaggggcg actatagcca caccttctcc
     4621 cgcaaggatc tgcaggccga gctgatcctg ctgttcgaga agcagaagga gtttggcaat
     4681 ccacacgtga gcggaggcct gaaggaggga atcgagaccc tgctgatgac acagaggcct
     4741 gccctgtccg gcgacgcagt gcagaagatg ctgggacact gcaccttcga gcctgcagag
     4801 ccaaaggccg ccaagaacac ctacacagcc gagcggttta tctggctgac aaagctgaac
     4861 aatctgagaa tcctggagca gggatccgag aggccactga ccgacacaga gagggccacc
     4921 ctgatggatg agccttaccg gaagtctaag ctgacatatg cccaggccag aaagctgctg
     4981 ggcctggagg acaccgcctt ctttaagggc ctgagatacg gcaaggataa tgccgaggcc
     5041 tccacactga tggagatgaa ggcctatcac gccatctctc gcgccctgga gaaggagggc
     5101 ctgaaggaca agaagtcccc cctgaacctg agctccgagc tgcaggatga gatcggcacc
     5161 gccttctctc tgtttaagac cgacgaggat atcacaggcc gcctgaagga cagggtgcag
     5221 cctgagatcc tggaggccct gctgaagcac atctctttcg ataagtttgt gcagatcagc
     5281 ctgaaggccc tgagaaggat cgtgccactg atggagcagg gcaagcggta cgacgaggcc
     5341 tgcgccgaga tctacggcga tcactatggc aagaagaaca cagaggagaa gatctatctg
     5401 ccccctatcc ctgccgacga gatcagaaat cctgtggtgc tgagggccct gtcccaggca
     5461 agaaaagtga tcaacggagt ggtgcgccgg tacggatctc cagcccggat ccacatcgag
     5521 accgccagag aagtgggcaa gagcttcaag gaccggaagg agatcgagaa gagacaggag
     5581 gagaatcgca aggatcggga gaaggccgcc gccaagttta gggagtactt ccctaacttt
     5641 gtgggcgagc caaagtctaa ggacatcctg aagctgcgcc tgtacgagca gcagcacggc
     5701 aagtgtctgt atagcggcaa ggagatcaat ctggtgcggc tgaacgagaa gggctatgtg
     5761 gagatcgatc acgccctgcc tttctccaga acctgggacg attcttttaa caataaggtg
     5821 ctggtgctgg gcagcgagaa ccagaataag ggcaatcaga caccatacga gtatttcaat
     5881 ggcaaggaca actccaggga gtggcaggag ttcaaggccc gcgtggagac ctctagattt
     5941 cccaggagca agaagcagcg gatcctgctg cagaagttcg acgaggatgg ctttaaggag
     6001 tgcaacctga atgacaccag atacgtgaac cggttcctgt gccagtttgt ggccgatcac
     6061 atcctgctga ccggcaaggg caagagaagg gtgttcgcct ctaatggcca gatcacaaac
     6121 ctgctgaggg gattttgggg actgaggaag gtgcgggcag agaatgacag acaccacgca
     6181 ctggatgcag tggtggtggc atgcagcacc gtggcaatgc agcagaagat cacaagattc
     6241 gtgaggtata aggagatgaa cgcctttgac ggcaagacca tcgataagga gacaggcaag
     6301 gtgctgcacc agaagaccca cttcccccag ccttgggagt tctttgccca ggaagtgatg
     6361 atccgggtgt tcggcaagcc agacggcaag cctgagtttg aggaggccga taccccagag
     6421 aagctgagga cactgctggc agagaagctg tctagcaggc cagaggcagt gcacgagtac
     6481 gtgaccccac tgttcgtgtc cagggcaccc aatcggaaga tgtctggcgc ccacaaggac
     6541 acactgagaa gcgccaagag gtttgtgaag cacaacgaga agatctccgt gaagagagtg
     6601 tggctgaccg agatcaagct ggccgatctg gagaacatgg tgaattacaa gaacggcagg
     6661 gagatcgagc tgtatgaggc cctgaaggca aggctggagg cctacggagg aaatgccaag
     6721 caggccttcg acccaaagga taaccccttt tataagaagg gaggacagct ggtgaaggcc
     6781 gtgcgggtgg agaagaccca ggagagcggc gtgctgctga ataagaagaa cgcctacaca
     6841 atcgccgaca atggcgatat ggtgagagtg gacgtgttct gtaaggtgga taagaagggc
     6901 aagaatcagt actttatcgt gcctatctat gcctggcagg tggccgagaa catcctgcca
     6961 gacatcgatt gcaagggcta cagaatcgac gatagctata cattctgttt ttccctgcac
     7021 aagtatgacc tgatcgcctt ccagaaggat gagaagtcca aggtggagtt tgcctactat
     7081 atcaattgcg actcctctaa cggcaggttc tacctggcct ggcacgataa gggcagcaag
     7141 gagcagcagt ttcgcatctc cacccagaat ctggtgctga tccagaagta tcaggtgaac
     7201 gagctgggca aggagatcag gccatgtcgg ctgaagaagc gcccacccgt gtctggcggc
     7261 tcaaaaagaa ccgccgacgg cagcgaattc gagcccaaga agaagaggaa agtctaatag
     7321 atctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     7381 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     7441 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     7501 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggctcgagcg
     7561 gcccaagctt aaaaaaataa acgatgcccc ttaaagcaga agctttaagg ggcagagcgt
     7621 tgcggcacat cttttcagac ggccttattg tagcaacggt tctcatttcg tttccgaaat
     7681 gagaaaggga gctacaaccg agacgcatta atcgtctcgg gtgtttcgtc ctttccacaa
     7741 gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat
     7801 tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt
     7861 attttgtact aatatctttg tgtttacagt caaattaatt ctaattatct ctctaacagc
     7921 cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctctt cctgcccgac
     7981 cttgcggccg c
//
