LOCUS       sequence-455739-        6725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TadA8e and Sauri cas9N by MYL2 promoter and sgRNA 
            targeting murine Camk2d intron7 5' splice site by U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6725)
  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 6725)
  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-MYL2-HA-TadA8e-Sauri N
            aa1-438-inteinN-U6...
FEATURES             Location/Qualifiers
     source          1..6725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   12..141
                     /label=AAV2 ITR
     regulatory      559..568
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             571..597
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             598..618
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2847..3071
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3203..3451)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3261..3280)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3431..3451)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   3786..3915
                     /label=AAV2 ITR
     primer_bind     complement(3934..3951)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3934..3950)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3943..3965)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4092..4547
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4179..4198)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4389..4410
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4573..4677
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4678..4746,4747..5538)
                     /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(4896..4915)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5709..6297
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6198..6217
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6585..6606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6621..6626,6627..6644,6645..6651)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6659..6675
                     /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     6664..6686
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6683..6699
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6683..6699
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ccttaattag gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacgt
      181 agccatgctc taggaagatc ggaattcgga gttccggggt tactcttccc cattagacaa
      241 tggcagggaa gagagcacac cccatcatcc ccaggccagg ccccagccac tgactcttta
      301 accttgaagg catttttggg tctcacgtgt ccacccaggc gggtggccgc ctttgagcag
      361 ctcttacttc agaagaacgg catggagtgg ggggtggggg gcttaggtgg cctccgcctc
      421 acctacaact gccaaaagtg gtcatggggt tatttttaac cccaggggag aggtatttat
      481 tgttccacag caggggcaga ggccagcagg ctcctcgaac tctccagagg tggcaactgg
      541 cctcagacac cgaccggtgc caccatggcc tacccatacg atgttccaga ttacgctcca
      601 aagaagaagc ggaaagtctc tgaggtggag ttttcccacg agtactggat gagacatgcc
      661 ctgaccctgg ccaagagggc acgggatgag agggaggtgc ctgtgggagc cgtgctggtg
      721 ctgaacaata gagtgatcgg cgagggctgg aacagagcca tcggcctgca cgacccaaca
      781 gcccatgccg aaattatggc cctgagacag ggcggcctgg tcatgcagaa ctacagactg
      841 attgacgcca ccctgtacgt gacattcgag ccttgcgtga tgtgcgccgg cgccatgatc
      901 cactctagga tcggccgcgt ggtgtttggc gtgaggaact caaaaagagg cgccgcaggc
      961 tccctgatga acgtgctgaa ctaccccggc atgaatcacc gcgtcgaaat taccgaggga
     1021 atcctggcag atgaatgtgc cgccctgctg tgcgatttct atcggatgcc tagacaggtg
     1081 ttcaatgctc agaagaaggc ccagagctcc atcaactccg gaggatctag cggaggctcc
     1141 tctggctctg agacacctgg cacaagcgag agcgcaacac ctgaaagcag cgggggcagc
     1201 agcggggggt caatgcagga gaaccagcag aagcaaaatt acatcctggg cctggccatc
     1261 ggcatcacca gcgtgggcta tggcctgatc gacagcaaga ccagagaagt gattgacgcc
     1321 ggcgtgcggc tattcccaga ggccgactct gaaaacaaca gcaatagaag atctaagcgg
     1381 ggcgcccgga gactgaaaag acgaagaatc cacagactga acagagtgaa agacctcctg
     1441 gctgactacc agatgatcga cttaaacaac gtgcccaagt ctaccgaccc ctacaccatc
     1501 cgggtgaagg gactgcggga acctctgacc aaggaagagt ttgccatcgc tctgctgcat
     1561 atcgccaaga gaagaggcct gcacaacatc tccgtgagca tgggcgatga ggaacaggac
     1621 aacgagctgt ccaccaagca gcagctgcag aagaacgctc agcagctgca ggacaaatac
     1681 gtgtgcgagc tgcaactgga aagactgacc aacatcaaca aggtcagagg cgagaagaac
     1741 cggttcaaga ccgaagattt cgtgaaggaa gtgaagcagc tgtgcgagac ccagcggcag
     1801 taccacaaca tcgacgatca gttcatccag cagtacatcg acctggtgag cacccggaga
     1861 gaatacttcg agggccctgg caacggctct ccatatggct gggatggaga tctgctgaag
     1921 tggtatgaga agctgatggg cagatgcacc tacttccctg aggagctgag aagcgtgaag
     1981 tacgcctaca gcgccgatct gtttaacgcc ctgaacgatc tgaacaacct ggttgtgacc
     2041 cgcgacgaca accctaagct ggaatactac gagaaatacc acattatcga gaacgtgttc
     2101 aagcagaaga aaaatccaac tctgaagcaa atcgccaaag agatcggcgt gcaggattac
     2161 gacatcagag gatacagaat taccaagtcc ggtaagcctc agttcaccag cttcaaactc
     2221 taccacgacc tgaaaaacat ctttgaacag gccaaatacc tggaagatgt ggagatgctg
     2281 gatgagatag ctaaaatcct gacaatctac caagacgaga tcagcatcaa gaaagccctg
     2341 gaccagctgc ctgagctgct gaccgagagc gaaaaaagcc agatcgctca gctgaccggc
     2401 tacaccggta cacatagact gtctctgaag tgcatccaca tcgtgatcga cgagctgtgg
     2461 gagagccccg aaaaccagat ggaaatcttc accagactga acctcaagcc aaagaaggtg
     2521 gaaatgtgtc tggctggcga tactctcatt accctggccg atggacgacg agtgcctatt
     2581 agagaactgg tgtcacagca gaatttttcc gtgtgggctc tgaatcctca gacttaccgc
     2641 ctggagaggg ctagagtgag tagagctttc tgtaccggca tcaaacctgt gtaccgcctc
     2701 accactagac tggggagatc cattagggcc actgccaacc accgatttct cacacctcag
     2761 ggctggaaac gagtcgatga actccagcct ggagattacc tggctctgcc taggagaatc
     2821 cctactgcct cctgaaccgg ttggctctgt gccttctagt tgccagccat ctgttgtttg
     2881 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2941 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3001 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3061 gggctctatg gctcgagcgg cccaagctta aaaaaatctc gccaacaagt tgacgagata
     3121 aacacggcat tttgccttgt tttagtagat tctgtaattt tcattacaga gtactaaaac
     3181 caggcatggt ttggtaagta gcggtgtttc gtcctttcca caagatatat aaagccaaga
     3241 aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa cataatttta
     3301 aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt actaatatct
     3361 ttgtgtttac agtcaaatta attccaatta tctctctaac agccttgtat cgtatatgca
     3421 aatatgaagg aatcatggga aataggccct cggtaccggc gatgctggct gccatgaaca
     3481 aaggttggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat
     3541 tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt
     3601 tttctttaac atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct
     3661 cctgactact cccagtcata gctgtccctc ttctcttatg gagatcggat ccgaattccc
     3721 gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa
     3781 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3841 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3901 cgagcgagcg cgcagcctta attaacctaa ttcactggcc gtcgttttac aacgtcgtga
     3961 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4021 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4081 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4141 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4201 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4261 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4321 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4381 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4441 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4501 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     4561 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4621 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4681 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4741 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4801 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4861 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4921 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4981 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5041 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5101 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5161 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5221 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5281 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5341 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5401 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5461 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5521 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5581 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5641 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5701 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5761 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5821 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5881 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5941 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6001 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6061 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6121 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6181 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6241 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6301 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6361 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6421 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     6481 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     6541 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     6601 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     6661 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc cagatttaat
     6721 taagg
//
