LOCUS       sequence-443751-        6023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing the Zfr/eGFP cassette. Translation of GFP is 
            controlled by splicing modulation of the Zfr by risdiplam..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6023)
  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 6023)
  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-CMV-Zf-GFP
FEATURES             Location/Qualifiers
     source          1..6023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(250..252,253..966)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(250..252,253..963)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(292..313)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(553..572)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     900..921
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1040..1059)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1105..1160
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   1253..1382
                     /label=AAV2 ITR
     primer_bind     complement(1401..1418)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1401..1417)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1410..1432)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1559..2014
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1646..1665)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1856..1877
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2040..2144
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2145..2213,2214..3005)
                     /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(2363..2382)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3176..3764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3665..3684
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4052..4073
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4088..4093,4094..4111,4112..4118)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4126..4142
                     /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     4131..4153
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4150..4166
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4150..4166
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4204..4333
                     /label=AAV2 ITR
     enhancer        4420..4799
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4800..5003
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4954..4974
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 acatggagaa gttaacttag taagaattga tgtgcttctg tgccagtgag atagctctgc
       61 aggtaaaagg gagactgacc cccaacttgt actctggccg tggcagtcat gtgcacacaa
      121 gtaaactgaa taaatataaa gagcagattt tagagtgttt tggctttaaa ttaattctat
      181 attctaactc tatttgcagt ttgcattgag actccttgca ttccgtcaga tacacaaagt
      241 tgcaagcttg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      301 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      361 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      421 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
      481 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      541 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
      601 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
      661 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
      721 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
      781 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
      841 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
      901 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
      961 aagtaataag ctcgcgtggt acctctagag tcgacccggg cggcctcgag gacggggtga
     1021 actacgcctg aggatccgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc
     1081 cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt
     1141 ggaatttttt gtgtctctca ctcggaagca attcgttgat ctgaatttcg accacccata
     1201 atacccatta ccctggtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc
     1261 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     1321 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     1381 agccttaatt aacctaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     1441 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     1501 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     1561 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1621 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1681 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1741 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1801 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1861 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1921 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1981 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     2041 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2101 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2161 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2221 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2281 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2341 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     2401 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2461 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2521 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2581 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2641 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     2701 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2761 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2821 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2881 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2941 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3001 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3061 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3121 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3181 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3241 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3301 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     3361 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3421 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3481 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3541 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3601 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3661 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3721 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3781 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3841 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3901 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     3961 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     4021 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     4081 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     4141 aatttcacac aggaaacagc tatgaccatg attacgccag atttaattaa ggccttaatt
     4201 aggctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
     4261 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
     4321 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac cagggtaatg
     4381 gggatcctct agaactatag ctagccagat atacgcgttg acattgatta ttgactagtt
     4441 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     4501 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     4561 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     4621 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     4681 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     4741 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     4801 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     4861 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     4921 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     4981 gggaggtcta tataagcaga gctcctcgag aagcggccgc actactccgc actgaccaac
     5041 agcgtgaaga cattacatcc agtgcacagg taattgaatt cttgttggat tactgtctct
     5101 aaggcctggg agttttataa ttgtttcttt atgtgtaata ataaccgtac tagtcattgt
     5161 gatacttatt tgggaagtaa gcctttaaag aggttattaa aaagatttac tgattcccac
     5221 accggaagtt gctcttctag ccgtgaaaga aggacggcgt ggtcttcagt ttgctatgtc
     5281 cactaacaat atgtccgacc cacgaaggcc caccaaagtt atacttgtgg cacatataaa
     5341 cctttggaat caagggacct gatactggat tattaaatat ttaagagaat ttatatttaa
     5401 taaacttgat acttcagtga gaaaggacaa atcatcagta gaatggtttt gagatagctg
     5461 atgattctat taaaagtaga tatgtgtgtg ggttaaatga cttaagcata catcaaaaga
     5521 ctgagaaagg ctcttaacat taaaagatgg gccttttttt ttcttttagg ttaaaggatt
     5581 acatattaaa aatattgtat agactcttta aggccaaaga agtgtgctca tttctcccga
     5641 ttaaaagtta tgagtaagaa ttgggtattt tgccttttgg gggtgggaga tttgaaacag
     5701 ggtttctcct tacagctctg gctttcctgg agcagtgaat tgagcctggc tcctctgtag
     5761 gagtaacgag ttgctttaac cactgacatc tttccaccac caccaccacc ccttatgaat
     5821 agactaaatt tatttaaaga ttgatatttt ctagcttaat tatttggaaa gcaaataagc
     5881 acacagaaaa gcctcctccc caaaaaaata taaataaaaa agtctatcag gacatagtat
     5941 atgtcccttc tgattaatca ttggtggtga aacaacccat tcagggtgtc tcttccatca
     6001 cagtggattt ttttcatatt gtt
//
