LOCUS       sequence-455603-        6542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Sp cas9C 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 6542)
  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 6542)
  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-inteinC-SpC aa714-1368-U6-Camk2d sgRNA7
FEATURES             Location/Qualifiers
     source          1..6542
                     /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"
     regulatory      558..567
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2723..2743
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2758..2813
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2812..2831)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     misc_RNA        complement(2918..2993)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3015..3263)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3073..3092)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3243..3263)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   3598..3727
                     /label=AAV2 ITR
     primer_bind     complement(3746..3763)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3746..3762)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3755..3777)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3904..4359
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3991..4010)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4201..4222
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4385..4489
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4490..4558,4559..5350)
                     /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(4708..4727)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5521..6109
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6010..6029
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6397..6418
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6433..6438,6439..6456,6457..6463)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6471..6487
                     /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     6476..6498
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6495..6511
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6495..6511
                     /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 tggcggcggc gtgcccggaa ctgcgtcagc tggcgcagag
      601 cgatgtgtat tgggatccga ttgtgagcat tgaaccggat ggcgtggaag aagtgtttga
      661 tctgaccgtg ccgggcccgc ataactttgt ggcgaacgat attattgcgc ataactccgg
      721 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
      781 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
      841 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
      901 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
      961 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     1021 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     1081 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     1141 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
     1201 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     1261 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     1321 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     1381 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     1441 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     1501 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     1561 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
     1621 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     1681 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     1741 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     1801 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     1861 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     1921 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     1981 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     2041 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     2101 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     2161 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     2221 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     2281 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     2341 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     2401 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     2461 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     2521 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
     2581 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     2641 gtacgagaca cggatcgacc tgtctcagct gggaggcgac tctggcggct caaaaagaac
     2701 cgccgacggc agcgaattcg agcccaagaa gaagaggaaa gtctaaaccg gttggctaat
     2761 aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa
     2821 ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg
     2881 caacatatgc ccatatgagg ccctcacgcg taaaaaagca ccgactcggt gccacttttt
     2941 caagttgata acggactagc cttattttaa cttgctattt ctagctctaa aacgcaggca
     3001 tggtttggta agtcggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa
     3061 tactttcaag ttacggtaag catatgatag tccattttaa aacataattt taaaactgca
     3121 aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt
     3181 acagtcaaat taattccaat tatctctcta acagccttgt atcgtatatg caaatatgaa
     3241 ggaatcatgg gaaataggcc ctcggtaccg gcgatgctgg ctgccatgaa caaaggttgg
     3301 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga
     3361 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta
     3421 acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta
     3481 ctcccagtca tagctgtccc tcttctctta tggagatcgg atccgaattc ccgataagga
     3541 tcttcctaga gcatggctac gtagataagt agcatggcgg gttaatcatt aactacaagg
     3601 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     3661 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     3721 cgcgcagcct taattaacct aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     3781 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     3841 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     3901 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3961 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4021 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4081 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     4141 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4201 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     4261 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4321 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     4381 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4441 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4501 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4561 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4621 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4681 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     4741 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     4801 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     4861 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4921 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4981 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     5041 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     5101 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5161 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     5221 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5281 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5341 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     5401 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5461 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5521 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5581 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5641 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5701 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5761 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5821 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5881 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5941 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6001 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6061 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6121 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6181 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     6241 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     6301 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     6361 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     6421 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     6481 ataacaattt cacacaggaa acagctatga ccatgattac gccagattta attaaggcct
     6541 ta
//
