LOCUS       sequence-443753-        4812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A multicloning site (MCS) was synthesized and inserted into 
            AAV-CMV-GFP to construct the AAV-CMV-MCS-(no ATG)-GFP negative 
            control plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4812)
  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 4812)
  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-MCS-noATG-GFP
FEATURES             Location/Qualifiers
     source          1..4812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     210..231
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(350..369)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    415..470
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   563..692
                     /label=AAV2 ITR
     primer_bind     complement(711..728)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(711..727)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(720..742)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      869..1324
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(956..975)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1166..1187
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1350..1454
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1455..1523,1524..2315)
                     /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(1673..1692)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2486..3074
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2975..2994
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3362..3383
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3398..3403,3404..3421,3422..3428)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3436..3452
                     /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     3441..3463
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3460..3476
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3460..3476
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   3514..3643
                     /label=AAV2 ITR
     enhancer        3730..4109
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4110..4313
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4264..4284
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(4372..4374,4375..4812,1..276)
                     /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(4372..4374,4375..4812,1..273)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4414..4435)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4675..4694)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
       61 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
      121 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
      181 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
      241 gggatcactc tcggcatgga cgagctgtac aagtaataag ctcgcgtggt acctctagag
      301 tcgacccggg cggcctcgag gacggggtga actacgcctg aggatccgat ctttttccct
      361 ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa
      421 ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagca
      481 attcgttgat ctgaatttcg accacccata atacccatta ccctggtaga taagtagcat
      541 ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg
      601 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
      661 cgggcggcct cagtgagcga gcgagcgcgc agccttaatt aacctaattc actggccgtc
      721 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
      781 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
      841 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
      901 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
      961 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1021 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1081 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1141 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1201 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1261 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     1321 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1381 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1441 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1501 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1561 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1621 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1681 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1741 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1801 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1861 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1921 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1981 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     2041 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2101 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2161 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2221 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2281 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2341 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2401 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2461 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2521 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2581 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2641 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2701 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2761 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2821 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2881 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2941 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3001 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3061 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3121 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3181 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3241 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     3301 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     3361 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     3421 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     3481 attacgccag atttaattaa ggccttaatt aggctgcgcg ctcgctcgct cactgaggcc
     3541 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
     3601 gcgcgcagag agggagtggc caactccatc actaggggtt ccttgtagtt aatgattaac
     3661 ccgccatgct acttatctac cagggtaatg gggatcctct agaactatag ctagccagat
     3721 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     3781 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     3841 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     3901 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     3961 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     4021 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     4081 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     4141 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     4201 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     4261 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcctcgag
     4321 aagcggccgc actactccgc ggactactac tagtttcacg aattcaagct tgtgagcaag
     4381 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     4441 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     4501 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     4561 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     4621 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     4681 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     4741 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     4801 aactacaaca gc
//
