LOCUS       sequence-455597-        7475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TadA8e and Sp 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 7475)
  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 7475)
  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-TadA8e-SpN aa2-713- inteinN-U6-Camk2...
FEATURES             Location/Qualifiers
     source          1..7475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   7..136
                     /label=AAV2 ITR
     regulatory      554..563
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             566..586
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1181..2896
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     primer_bind     complement(3636..3655)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3665..3746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3690..3709
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_RNA        complement(3851..3926)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3948..4196)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4006..4025)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4176..4196)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4531..4660
                     /label=AAV2 ITR
     primer_bind     complement(4679..4696)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4679..4695)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4688..4710)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4837..5292
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4924..4943)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5134..5155
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5318..5422
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5423..5491,5492..6283)
                     /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(5641..5660)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6454..7042
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6943..6962
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7330..7351
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7366..7371,7372..7389,7390..7396)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7404..7420
                     /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     7409..7431
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7428..7444
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7428..7444
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 attaggctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
       61 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc
      121 atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca
      181 tgctctagga agatcggaat tcggagttcc ggggttactc ttccccatta gacaatggca
      241 gggaagagag cacaccccat catccccagg ccaggcccca gccactgact ctttaacctt
      301 gaaggcattt ttgggtctca cgtgtccacc caggcgggtg gccgcctttg agcagctctt
      361 acttcagaag aacggcatgg agtggggggt ggggggctta ggtggcctcc gcctcaccta
      421 caactgccaa aagtggtcat ggggttattt ttaaccccag gggagaggta tttattgttc
      481 cacagcaggg gcagaggcca gcaggctcct cgaactctcc agaggtggca actggcctca
      541 gacaccgacc ggtgccacca tggccccaaa gaagaagcgg aaagtctctg aggtggagtt
      601 ttcccacgag tactggatga gacatgccct gaccctggcc aagagggcac gggatgagag
      661 ggaggtgcct gtgggagccg tgctggtgct gaacaataga gtgatcggcg agggctggaa
      721 cagagccatc ggcctgcacg acccaacagc ccatgccgaa attatggccc tgagacaggg
      781 cggcctggtc atgcagaact acagactgat tgacgccacc ctgtacgtga cattcgagcc
      841 ttgcgtgatg tgcgccggcg ccatgatcca ctctaggatc ggccgcgtgg tgtttggcgt
      901 gaggaactca aaaagaggcg ccgcaggctc cctgatgaac gtgctgaact accccggcat
      961 gaatcaccgc gtcgaaatta ccgagggaat cctggcagat gaatgtgccg ccctgctgtg
     1021 cgatttctat cggatgccta gacaggtgtt caatgctcag aagaaggccc agagctccat
     1081 caactccgga ggatctagcg gaggctcctc tggctctgag acacctggca caagcgagag
     1141 cgcaacacct gaaagcagcg ggggcagcag cggggggtca gacaagaagt acagcatcgg
     1201 cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     1261 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     1321 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     1381 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     1441 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     1501 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     1561 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     1621 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     1681 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     1741 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     1801 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     1861 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     1921 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     1981 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     2041 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     2101 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     2161 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     2221 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     2281 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     2341 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     2401 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     2461 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     2521 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     2581 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     2641 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     2701 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     2761 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     2821 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     2881 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     2941 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     3001 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     3061 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     3121 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     3181 cggcatccga gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt
     3241 tgccaaccgg aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat
     3301 ccagaaagca caagtttgtc tggctggcga tactctcatt accctggccg atggacgacg
     3361 agtgcctatt agagaactgg tgtcacagca gaatttttcc gtgtgggctc tgaatcctca
     3421 gacttaccgc ctggagaggg ctagagtgag tagagctttc tgtaccggca tcaaacctgt
     3481 gtaccgcctc accactagac tggggagatc cattagggcc actgccaacc accgatttct
     3541 cacacctcag ggctggaaac gagtcgatga actccagcct ggagattacc tggctctgcc
     3601 taggagaatc cctactgcct cctgatctcg agctcgatga gtttggacaa accacaacta
     3661 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     3721 ccattataag ctgcaataaa caagttctcg gaaggacata tgggagggca aatcatttaa
     3781 aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg aggccctcac
     3841 gcgtaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt
     3901 taacttgcta tttctagctc taaaacgcag gcatggtttg gtaagtcggt gtttcgtcct
     3961 ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt aagcatatga
     4021 tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta ttactttcta
     4081 cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattcc aattatctct
     4141 ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag gccctcggta
     4201 ccggcgatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa
     4261 cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt
     4321 tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt
     4381 ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc
     4441 ttatggagat cggatccgaa ttcccgataa ggatcttcct agagcatggc tacgtagata
     4501 agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc
     4561 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4621 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ccttaattaa cctaattcac
     4681 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     4741 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     4801 cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa
     4861 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4921 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4981 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5041 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5101 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5161 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     5221 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5281 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5341 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5401 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5461 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5521 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5581 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5641 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5701 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5761 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5821 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5881 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5941 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6001 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6061 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6121 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6181 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6241 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6301 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6361 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6421 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6481 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6541 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6601 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6661 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6721 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6781 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6841 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6901 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6961 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7021 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7081 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     7141 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     7201 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     7261 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     7321 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     7381 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     7441 tgaccatgat tacgccagat ttaattaagg cctta
//
