LOCUS       sequence-326319-        7648 bp    DNA     circular SYN 14-APR-2025
DEFINITION  AAV-based delivery of ACTN2-GFP into cardiomyocytes in vivo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7648)
  AUTHORS   Guo Y, Cao Y, Jardin BD, Sethi I, Ma Q, Moghadaszadeh B, Troiano EC,
            Mazumdar N, Trembley MA, Small EM, Yuan GC, Beggs AH, Pu WT
  TITLE     Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF
            signaling.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Jan 12;118(2). pii: 2008861118. doi: 
            10.1073/pnas.2008861118.
  PUBMED    33361330
REFERENCE   2  (bases 1 to 7648)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2021-01-12"; volume: "118(2). pii: 
            2008861118. doi"; pages: " 10.1073/pnas.2008861118"
COMMENT     Sequence Label: aav-tnt-actn2-gfp
FEATURES             Location/Qualifiers
     source          1..7648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     intron          772..904
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     887..906
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        949..967
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3686..3695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3692..3694,3695..3697,3698..4411)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3692..3694,3695..3697,3698..4408)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3737..3758)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3998..4017)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4345..4366
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4485..4504)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4550..4605
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   4698..4827
                     /label=AAV2 ITR
     primer_bind     complement(4846..4863)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4846..4862)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4855..4877)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5004..5459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5091..5110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5301..5322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5485..5589
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5590..5658,5659..6450)
                     /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(5808..5827)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6621..7209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7110..7129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7497..7518
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7533..7538,7539..7556,7557..7563)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7571..7587
                     /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     7576..7598
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7595..7611
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7595..7611
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg
      181 atcctctaga actatagcta gaattcgccc ttacgggccc cccctcgagg tcgggataaa
      241 agcagtctgg gctttcacat gacagcatct ggggctgcgg cagagggtcg ggtccgaagc
      301 gctgccttat cagcgtcccc agccctggga ggtgacagct ggctggcttg tgtcagcccc
      361 tcgggcactc acgtatctcc gtccgacggg tttaaaatag caaaactctg aggccacaca
      421 atagcttggg cttatatggg ctcctgtggg ggaaggggga gcacggaggg ggccggggcc
      481 gctgctgcca aaatagcagc tcacaagtgt tgcattcctc tctgggcgcc gggcacattc
      541 ctgctggctc tgcccgcccc ggggtgggcg ccggggggac cttaaagcct ctgcccccca
      601 aggagccctt cccagacagc cgccggcacc caccgctccg tgggacgatc cccgaagctc
      661 tagagcttta ttgcggtagt ttatcacagt taaattgcta acgcagtcag tgcttctgac
      721 acaacagtct cgaacttaag ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc
      781 aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag acagagaaga
      841 ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc ctttctctcc
      901 acaggtgtcc actcccagtt caattacagc tcttaaggct agagtactta atacgactca
      961 ctataggcta gcctcgagaa gcggccgcat gaatcagata gagccaggag tacaatataa
     1021 ttacgtatac gacgaagatg agtatatgat ccaggaggag gagtgggatc gagatctact
     1081 actagatcca gcctgggaga agcagcagag gaagacgttc actgcctggt gcaactcaca
     1141 cctgcggaaa gctggcaccc agatcgagaa catcgaggag gatttcagga acggcctcaa
     1201 gctcatgcta cttctagaag tcatctcagg ggaaaggctg cccaaacctg accggggaaa
     1261 aatgcggttc cacaagattg cgaatgtcaa caaggctctg gattacatag ccagcaaggg
     1321 agtgaagctg gtatctattg gagctgaaga gattgttgac ggcaatgtga agatgacact
     1381 aggtatgata tggacaataa tcctccgctt cgccattcag gacatctcag tggaagaaac
     1441 atctgccaaa gaagggctgc tgctatggtg tcagaggaag acagctccat acagaaatgt
     1501 gaatatccag aacttccaca ccagctggaa agatggcctt ggactctgtg ccctcatcca
     1561 cagacaccgg cccgacctca ttgactactc aaagcttaac aaggatgacc ccataggaaa
     1621 cattaacctg gccatggaaa tagcagagaa gcacttggat atccctaaga tgttggatgc
     1681 tgaagacatt gtcaacactc ccaaacccga tgaaagagcc atcatgactt acgtttcctg
     1741 cttctaccat gctttcgcgg gagcggagca ggccgagaca gcagctaaca gaatatgtaa
     1801 ggttcttgct gtgaatcaag agaatgagag gctgatggaa gaatatgaga ggctagcgag
     1861 tgagctgttg gagtggattc ggcgcacaat cccctggctg gagaacagga cgcctgagaa
     1921 gaccatgcag gccatgcaga agaagctgga ggacttccgg gattaccgtc gtaagcataa
     1981 gccccccaag gtgcaggaga agtgccagct ggagatcaac ttcaacacgc tgcagaccaa
     2041 gcttcggatc agcaaccggc ccgccttcat gccctccgag ggcaagatgg tgtcggacat
     2101 tgctggtgcc tggcagaggt tggagcaggc tgagaagggc tatgaggagt ggctattgaa
     2161 tgagattcga agactggagc gcttggagca cttggctgag aagttcaggc agaaggcctc
     2221 cacgcacgag acatgggctt atggcaaaga acagatcttg ctgcagaagg actacgagtc
     2281 tgcgtccctg acggaagttc gagccctact tcggaagcac gaggcctttg agagcgacct
     2341 ggctgcacac caggaccgcg tggagcagat cgctgccatt gcgcaggagc tcaatgaact
     2401 ggactatcac gatgctgtga acgtcaatga tcggtgccag aaaatttgcg accagtggga
     2461 tagattgggg acgctcaccc agaagaggag ggaggccttg gagagaactg agaagttgct
     2521 ggaaacaatc gaccaactgc acctggagtt tgccaagaga gccgcgccct tcaacaactg
     2581 gatggaggga gccatggagg acctgcagga catgttcatt gtccacagca tcgaggagat
     2641 ccagagtctg atcactgcac atgagcagtt caaggccaca ctgcccgagg ccgacgggga
     2701 gcggcagtcc atcctggcca tccagaacga ggtggagaag gtgatccaga gctacagcat
     2761 ccggatcagc tccagcaacc cctacagcac tgtcaccatg gatgagctgc gcaacaagtg
     2821 ggacaaggtg aagcagctgg tgccggtccg ggatcagtcc ctgcaggagg agctggctcg
     2881 ccaacatgct aatgagcgtc tgcggcgcca gtttgctgct caggccaatg ccattgggcc
     2941 ctggatccag aacaagatgg aggagatcgc tcggagctcc atccagatca ccggggccct
     3001 ggaggaccag atgaaccagc tgaagcagta cgaacacaac atcatcaact acaagaacaa
     3061 catcgacaag ctggagggcg accaccagct catccaggag gccctggtct tcgacaacaa
     3121 gcacacgaac tacaccatgg agcatatccg tgtcggatgg gagttgctgc tgacaactat
     3181 cgccagaacc atcaatgagg tggagaccca gatcctgaca agagatgcca agggcatcac
     3241 ccaggagcag atgaatgagt tcagagcctc cttcaaccac tttgacagga ggaagaacgg
     3301 cctgatggat catgaggatt tcagagcctg cctcatttcc atgggctatg acttgggtga
     3361 agctgaattt gcccgcatta tgactttggt tgaccccaac ggacaaggca cggtcacctt
     3421 ccagtccttc attgacttca tgactagaga gaccgcagac accgacacgg cagagcaggt
     3481 catcgcctcc ttccggattc tggcttctga taagccttac atcttggcag aggagcttcg
     3541 tcgagagctg cctccggatc aggcccagta ctgcatcaag agaatgcccc catactcagg
     3601 cccgggcagt gtccccgggg ccctggacta cactgccttc tcctctgccc tctacgggga
     3661 aagcgacctc actagtttca cgcgggccgc catggtgagc aagggcgagg agctgttcac
     3721 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     3781 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     3841 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     3901 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     3961 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     4021 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     4081 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     4141 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     4201 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     4261 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     4321 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     4381 cactctcggc atggacgagc tgtacaagta ataagctcgc gtggtacctc tagagtcgac
     4441 ccgggcggcc tcgaggacgg ggtgaactac gcctgaggat ccgatctttt tccctctgcc
     4501 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     4561 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aagcaattcg
     4621 ttgatctgaa tttcgaccac ccataatacc cattaccctg gtagataagt agcatggcgg
     4681 gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4741 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4801 ggcctcagtg agcgagcgag cgcgcagcct taattaacct aattcactgg ccgtcgtttt
     4861 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     4921 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     4981 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     5041 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     5101 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5161 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     5221 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5281 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5341 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5401 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     5461 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5521 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5581 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5641 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5701 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5761 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5821 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5881 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5941 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6001 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6061 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6121 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6181 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6241 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6301 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6361 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6421 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6481 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6541 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6601 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6661 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6721 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6781 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6841 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6901 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6961 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7021 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7081 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7141 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7201 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7261 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     7321 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     7381 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     7441 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     7501 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     7561 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     7621 gccagattta attaaggcct taattagg
//
