LOCUS       sequence-443757-        7064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing the Zfr/Firefly luciferase/3xMIR122 cassette. 
            Translation of luciferase 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 7064)
  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 7064)
  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-luciferase-3xMIR122
FEATURES             Location/Qualifiers
     source          1..7064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(126..143)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(126..142)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(135..157)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      284..739
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(371..390)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     581..602
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        765..869
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(870..938,939..1730)
                     /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(1088..1107)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1901..2489
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2390..2409
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2777..2798
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2813..2818,2819..2836,2837..2843)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2851..2867
                     /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     2856..2878
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2875..2891
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2875..2891
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2929..3058
                     /label=AAV2 ITR
     CDS             4918..4974
                     /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"
     CDS             4975..5001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(5065..5083)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     primer_bind     complement(6829..6848)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6894..6949
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   join(7042..7064,1..107)
                     /label=AAV2 ITR
ORIGIN
        1 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
       61 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagcct taattaacct
      121 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
      181 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
      241 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
      301 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
      361 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
      421 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
      481 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
      541 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
      601 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
      661 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
      721 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
      781 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
      841 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
      901 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
      961 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1021 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1081 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1141 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1201 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1261 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     1321 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     1381 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     1441 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     1501 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     1561 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     1621 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     1681 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     1741 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     1801 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     1861 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     1921 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1981 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2041 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2101 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2161 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2221 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2281 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2341 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2401 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2461 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2521 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     2581 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     2641 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     2701 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2761 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     2821 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     2881 acagctatga ccatgattac gccagattta attaaggcct taattaggct gcgcgctcgc
     2941 tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc
     3001 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttccttg
     3061 tagttaatga ttaacccgcc atgctactta tctaccaggg taatggggat cctctagaac
     3121 tatagctcag tccattagga gccagtagcc tggaagatgt ctttaccccc agcatcagtt
     3181 caagtggagc agcacataac tcttgccctc tgccttccaa gattctggtg ctgagactta
     3241 tggagtgtct tggaggttgc cttctgcccc ccaaccctgc tcccagctgg ccctcccagg
     3301 cctgggttgc tggcctctgc tttatcagga ttctcaagag ggacagctgg tttatgttgc
     3361 atgactgttc cctgcatatc tgctctggtt ttaaatagct tatctgagca gctggaggac
     3421 cacatgggct tatatggcgt ggggtacatg ttcctgtagc cttgtccctg gcacctgcca
     3481 aaatagcagc caacaccccc cacccccacc gccatccccc tgccccaccc gtcccctgtc
     3541 gcacattcct ccctccgcag ggctggctca ccaggcccca gcccacatgc ctgcttaaag
     3601 ccctctccat cctctgcctc acccagtccc cgctgagact gagcagacgc ctccaggatc
     3661 tgtcggcaga gctcctcgag aagcactgac caacagcgtg aagacattac atccagtgca
     3721 caggtaattg aattcttgtt ggattactgt ctctaaggcc tgggagtttt ataattgttt
     3781 ctttatgtgt aataataacc gtactagtca ttgtgatact tatttgggaa gtaagccttt
     3841 aaagaggtta ttaaaaagat ttactgattc ccacaccgga agttgctctt ctagccgtga
     3901 aagaaggacg gcgtggtctt cagtttgcta tgtccactaa caatatgtcc gacccacgaa
     3961 ggcccaccaa agttatactt gtggcacata taaacctttg gaatcaaggg acctgatact
     4021 ggattattaa atatttaaga gaatttatat ttaataaact tgatacttca gtgagaaagg
     4081 acaaatcatc agtagaatgg ttttgagata gctgatgatt ctattaaaag tagatatgtg
     4141 tgtgggttaa atgacttaag catacatcaa aagactgaga aaggctctta acattaaaag
     4201 atgggccttt ttttttcttt taggttaaag gattacatat taaaaatatt gtatagactc
     4261 tttaaggcca aagaagtgtg ctcatttctc ccgattaaaa gttatgagta agaattgggt
     4321 attttgcctt ttgggggtgg gagatttgaa acagggtttc tccttacagc tctggctttc
     4381 ctggagcagt gaattgagcc tggctcctct gtaggagtaa cgagttgctt taaccactga
     4441 catctttcca ccaccaccac caccccttat gaatagacta aatttattta aagattgata
     4501 ttttctagct taattatttg gaaagcaaat aagcacacag aaaagcctcc tccccaaaaa
     4561 aatataaata aaaaagtcta tcaggacata gtatatgtcc cttctgatta atcattggtg
     4621 gtgaaacaac ccattcaggg tgtctcttcc atcacagtgg atttttttca tattgttaca
     4681 tggagaagtt aacttagtaa gaattgatgt gcttctgtgc cagtgagata gctctgcagg
     4741 taaaagggag actgaccccc aacttgtact ctggccgtgg cagtcatgtg cacacaagta
     4801 aactgaataa atataaagag cagattttag agtgttttgg ctttaaatta attctatatt
     4861 ctaactctat ttgcagtttg cattgagact ccttgcattc cgtcagatac acaaagtgct
     4921 actaacttca gcctactaaa acaggctgga gacgttgagg agaaccctgg accttatcca
     4981 tacgacgttc cagattacgc agaagacgcc aaaaacataa agaaaggccc ggcgccattc
     5041 tatccgctgg aagatggaac cgctggagag caactgcata aggctatgaa gagatacgcc
     5101 ctggttcctg gaacaattgc ttttacagat gcacatatcg aggtggacat cacttacgct
     5161 gagtacttcg aaatgtccgt tcggttggca gaagctatga aacgatatgg gctgaataca
     5221 aatcacagaa tcgtcgtatg cagtgaaaac tctcttcaat tctttatgcc ggtgttgggc
     5281 gcgttattta tcggagttgc agttgcgccc gcgaacgaca tttataatga acgtgaattg
     5341 ctcaacagta tgggcatttc gcagcctacc gtggtgttcg tttccaaaaa ggggttgcaa
     5401 aaaattttga acgtgcaaaa aaagctccca atcatccaaa aaattattat catggattct
     5461 aaaacggatt accagggatt tcagtcgatg tacacgttcg tcacatctca tctacctccc
     5521 ggttttaatg aatacgattt tgtgccagag tccttcgata gggacaagac aattgcactg
     5581 atcatgaact cctctggatc tactggtctg cctaaaggtg tcgctctgcc tcatagaact
     5641 gcctgcgtga gattctcgca tgccagagat cctatttttg gcaatcaaat cattccggat
     5701 actgcgattt taagtgttgt tccattccat cacggttttg gaatgtttac tacactcgga
     5761 tatttgatat gtggatttcg agtcgtctta atgtatagat ttgaagaaga gctgtttctg
     5821 aggagccttc aggattacaa gattcaaagt gcgctgctgg tgccaaccct attctccttc
     5881 ttcgccaaaa gcactctgat tgacaaatac gatttatcta atttacacga aattgcttct
     5941 ggtggcgctc ccctctctaa ggaagtcggg gaagcggttg ccaagaggtt ccatctgcca
     6001 ggtatcaggc aaggatatgg gctcactgag actacatcag ctattctgat tacacccgag
     6061 ggggatgata aaccgggcgc ggtcggtaaa gttgttccat tttttgaagc gaaggttgtg
     6121 gatctggata ccgggaaaac gctgggcgtt aatcaaagag gcgaactgtg tgtgagaggt
     6181 cctatgatta tgtccggtta tgtaaacaat ccggaagcga ccaacgcctt gattgacaag
     6241 gatggatggc tacattctgg agacatagct tactgggacg aagacgaaca cttcttcatc
     6301 gttgaccgcc tgaagtctct gattaagtac aaaggctatc aggtggctcc cgctgaattg
     6361 gaatccatct tgctccaaca ccccaacatc ttcgacgcag gtgtcgcagg tcttcccgac
     6421 gatgacgccg gtgaacttcc cgccgccgtt gttgttttgg agcacggaaa gacgatgacg
     6481 gaaaaagaga tcgtggatta cgtcgccagt caagtaacaa ccgcgaaaaa gttgcgcgga
     6541 ggagttgtgt ttgtggacga agtaccgaaa ggtcttaccg gaaaactcga cgcaagaaaa
     6601 atcagagaga tcctcataaa ggccaagaag ggcggaaaga tgccgtgtaa gtacctctag
     6661 aggatgggga ctgaaagctt cggtcaaaca ccattgtcac actccatact atcaaacacc
     6721 attgtcacac tccagatcaa caaacaccat tgtcacactc cacgccgaag cttatcgata
     6781 ccgcccgggc ggcctcgagg acggggtgaa ctacgcctga ggatccgatc tttttccctc
     6841 tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag
     6901 gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaagcaa
     6961 ttcgttgatc tgaatttcga ccacccataa tacccattac cctggtagat aagtagcatg
     7021 gcgggttaat cattaactac aaggaacccc tagtgatgga gttg
//
