LOCUS       sequence-443750-        6877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing the Zfr/YAP5SA/3xMIR122 cassette. Translation of 
            YAP5SA is controlled by splicing modulation of the Zfr specifically 
            in cardiomyocytes by risdiplam in vivo..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6877)
  AUTHORS   Chen Z, Yang L, Zhang Y, Li J, Yang Y, Li Y, Fan L, Chen W, Miao L, 
            Liu J, Chen G, Wang Z, Li Y, Gao F, Zhou J, Zheng L, Zhang Y, Zhao 
            D, Pu WT, Yang K, Dong E, Guo Y
  TITLE     The drug-elicitable alternative splicing module for tunable vector 
            expression in the heart.
  JOURNAL   Nat Cardiovasc Res. 2025 Jun 13. doi: 10.1038/s44161-025-00665-7.
  PUBMED    40514436
REFERENCE   2  (bases 1 to 6877)
  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: "Nat 
            Cardiovasc Res. 2025 Jun 13. doi: 10.1038/s44161-025-00665-7."
COMMENT     Sequence Label: pAAV-TNT4-Zfr-P2A-HA-YAP5SA-3xMIR122
FEATURES             Location/Qualifiers
     source          1..6877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             37..63
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1704..1723)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1769..1824
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   1917..2046
                     /label=AAV2 ITR
     primer_bind     complement(2065..2082)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2065..2081)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2074..2096)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2223..2678
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2310..2329)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2520..2541
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2704..2808
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2809..2877,2878..3669)
                     /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(3027..3046)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3840..4428
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4329..4348
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4716..4737
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4752..4757,4758..4775,4776..4782)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4790..4806
                     /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     4795..4817
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4814..4830
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4814..4830
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4868..4997
                     /label=AAV2 ITR
     CDS             join(6857..6877,1..36)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
ORIGIN
        1 aaacaggctg gagacgttga ggagaaccct ggaccttatc catacgacgt tccagattac
       61 gcagatcccg ggcagcagcc gccgcctcaa ccggcccccc agggccaagg gcagccgcct
      121 tcgcagcccc cgcaggggca gggcccgccg tccggacccg ggcaaccggc acccgcggcg
      181 acccaggcgg cgccgcaggc accccccgcc gggcatcaga tcgtgcacgt ccgcggggac
      241 gcggagaccg acctggaggc gctcttcaac gccgtcatga accccaagac ggccaacgtg
      301 ccccagaccg tgcccatgag gctccggaag ctgcccgact ccttcttcaa gccgccggag
      361 cccaaatccc actcccgaca ggccgctact gatgcaggca ctgcaggagc cctgactcca
      421 cagcatgttc gagctcatgc cgctccagct gctctgcagt tgggagctgt ttctcctggg
      481 acactgaccc ccactggagt agtctctggc ccagcagcta cacccacagc tcagcatctt
      541 cgacaggctg cttttgagat acctgatgat gtacctctgc cagcaggttg ggagatggca
      601 aagacatctt ctggtcagag atacttctta aatcacatcg atcagacaac aacatggcag
      661 gaccccagga aggccatgct gtcccagatg aacgtcacag cccccaccag tccaccagtg
      721 cagcagaata tgatgaactc ggcttcaggt cctcttcctg atggatggga acaagccatg
      781 actcaggatg gagaaattta ctatataaac cataagaaca agaccacctc ttggctagac
      841 ccaaggcttg accctcgttt tgccatgaac cagagaatca gtcagagtgc tccagtgaaa
      901 cagccaccac ccctggctcc ccagagccca cagggaggcg tcatgggtgg cagcaactcc
      961 aaccagcagc aacagatgcg actgcagcaa ctgcagatgg agaaggagag gctgcggctg
     1021 aaacagcaag aactgcttcg gcaggagtta gccctgcgta gccagttacc aacactggag
     1081 caggatggtg ggactcaaaa tccagtgtct tctcccggga tgtctcagga attgagaaca
     1141 atgacgacca atagctcaga tcctttcctt aacagtggca cctatcactc tcgagatgag
     1201 gctacagaca gtggactaag catgagcagc tacagtgtcc ctcgaacccc agatgacttc
     1261 ctgaacagtg tggatgagat ggatacaggt gatactatca accaaagcac cctgccctca
     1321 cagcagaacc gtttcccaga ctaccttgaa gccattcctg ggacaaatgt ggaccttgga
     1381 acactggaag gagatggaat gaacatagaa ggagaggagc tgatgccaag tctgcaggaa
     1441 gctttgagtt ctgacatcct taatgacatg gagtctgttt tggctgccac caagctagat
     1501 aaagaaagct ttcttacatg gttgtacctc taggtacgat ggggactgaa agcttcggtc
     1561 aaacaccatt gtcacactcc atactatcaa acaccattgt cacactccag atcaacaaac
     1621 accattgtca cactccacgc cgaagcttat cgataccgcc cgggcggcct cgaggacggg
     1681 gtgaactacg cctgaggatc cgatcttttt ccctctgcca aaaattatgg ggacatcatg
     1741 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     1801 tgttggaatt ttttgtgtct ctcactcgga agcaattcgt tgatctgaat ttcgaccacc
     1861 cataataccc attaccctgg tagataagta gcatggcggg ttaatcatta actacaagga
     1921 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     1981 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     2041 gcgcagcctt aattaaccta attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     2101 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     2161 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     2221 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2281 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2341 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2401 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2461 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2521 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2581 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2641 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     2701 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2761 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2821 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2881 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2941 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     3001 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     3061 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     3121 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     3181 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3241 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     3301 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     3361 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     3421 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     3481 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     3541 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3601 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3661 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3721 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3781 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3841 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3901 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3961 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     4021 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4081 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4141 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4201 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4261 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4321 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4381 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4441 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4501 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4561 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     4621 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     4681 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     4741 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     4801 taacaatttc acacaggaaa cagctatgac catgattacg ccagatttaa ttaaggcctt
     4861 aattaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc
     4921 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
     4981 catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctaccagggt
     5041 aatggggatc ctctagaact atagctcagt ccattaggag ccagtagcct ggaagatgtc
     5101 tttaccccca gcatcagttc aagtggagca gcacataact cttgccctct gccttccaag
     5161 attctggtgc tgagacttat ggagtgtctt ggaggttgcc ttctgccccc caaccctgct
     5221 cccagctggc cctcccaggc ctgggttgct ggcctctgct ttatcaggat tctcaagagg
     5281 gacagctggt ttatgttgca tgactgttcc ctgcatatct gctctggttt taaatagctt
     5341 atctgagcag ctggaggacc acatgggctt atatggcgtg gggtacatgt tcctgtagcc
     5401 ttgtccctgg cacctgccaa aatagcagcc aacacccccc acccccaccg ccatccccct
     5461 gccccacccg tcccctgtcg cacattcctc cctccgcagg gctggctcac caggccccag
     5521 cccacatgcc tgcttaaagc cctctccatc ctctgcctca cccagtcccc gctgagactg
     5581 agcagacgcc tccaggatct gtcggcagag ctcctcgaga agcactgacc aacagcgtga
     5641 agacattaca tccagtgcac aggtaattga attcttgttg gattactgtc tctaaggcct
     5701 gggagtttta taattgtttc tttatgtgta ataataaccg tactagtcat tgtgatactt
     5761 atttgggaag taagccttta aagaggttat taaaaagatt tactgattcc cacaccggaa
     5821 gttgctcttc tagccgtgaa agaaggacgg cgtggtcttc agtttgctat gtccactaac
     5881 aatatgtccg acccacgaag gcccaccaaa gttatacttg tggcacatat aaacctttgg
     5941 aatcaaggga cctgatactg gattattaaa tatttaagag aatttatatt taataaactt
     6001 gatacttcag tgagaaagga caaatcatca gtagaatggt tttgagatag ctgatgattc
     6061 tattaaaagt agatatgtgt gtgggttaaa tgacttaagc atacatcaaa agactgagaa
     6121 aggctcttaa cattaaaaga tgggcctttt tttttctttt aggttaaagg attacatatt
     6181 aaaaatattg tatagactct ttaaggccaa agaagtgtgc tcatttctcc cgattaaaag
     6241 ttatgagtaa gaattgggta ttttgccttt tgggggtggg agatttgaaa cagggtttct
     6301 ccttacagct ctggctttcc tggagcagtg aattgagcct ggctcctctg taggagtaac
     6361 gagttgcttt aaccactgac atctttccac caccaccacc accccttatg aatagactaa
     6421 atttatttaa agattgatat tttctagctt aattatttgg aaagcaaata agcacacaga
     6481 aaagcctcct ccccaaaaaa atataaataa aaaagtctat caggacatag tatatgtccc
     6541 ttctgattaa tcattggtgg tgaaacaacc cattcagggt gtctcttcca tcacagtgga
     6601 tttttttcat attgttacat ggagaagtta acttagtaag aattgatgtg cttctgtgcc
     6661 agtgagatag ctctgcaggt aaaagggaga ctgaccccca acttgtactc tggccgtggc
     6721 agtcatgtgc acacaagtaa actgaataaa tataaagagc agattttaga gtgttttggc
     6781 tttaaattaa ttctatattc taactctatt tgcagtttgc attgagactc cttgcattcc
     6841 gtcagataca caaagtgcta ctaacttcag cctacta
//
