LOCUS       sequence-422162-        7516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of cardiac troponin T.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7516)
  AUTHORS   Fetterman KA, Blancard M, Lyra-Leite DM, Vanoye CG, Fonoudi H, Jouni
            M, DeKeyser JL, Lenny B, Sapkota Y, George AL Jr, Burridge PW
  TITLE     Independent compartmentalization of functional, metabolic, and 
            transcriptional maturation of hiPSC-derived cardiomyocytes.
  JOURNAL   Cell Rep. 2024 May 28;43(5):114160. doi: 
            10.1016/j.celrep.2024.114160. Epub 2024 Apr 27.
  PUBMED    38678564
REFERENCE   2  (bases 1 to 7516)
  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: "Cell Rep.";
            date: "2024-05-28"; volume: "43(5):114160. doi"; pages: " 
            10.1016/j.celrep.2024.114160. Epub 2024 Apr 27"
COMMENT     Sequence Label: pAAVS1-Neo-TNNT2-Puro
FEATURES             Location/Qualifiers
     source          1..7516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    796..821
                     /label=SA
                     /note="splice acceptor site"
     CDS             845..898
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             908..1708
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="GTMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(968..987)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1578..1597
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1728..1745)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1734..1958
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             2611..3210
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2611..2630)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3107..3127
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3308..3327)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3373..3428
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3427..3446)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     misc_feature    3984..4513
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(4659..4678)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4660..4678)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4685..4702)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4685..4701)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4694..4716)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4843..5298
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4930..4949)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5140..5161
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5324..5428
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5429..5497,5498..6289)
                     /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(5647..5666)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6460..7048
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6949..6968
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7202..7219
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7336..7357
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7372..7377,7378..7395,7396..7402)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7410..7426
                     /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     7415..7437
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7434..7450
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7434..7450
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7471..7489
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(7502..7516,1..789)
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
ORIGIN
        1 gcattctctc ccctgggcct gtgccgcttt ctgtctgcag cttgtggcct gggtcacctc
       61 tacggctggc ccagatcctt ccctgccgcc tccttcaggt tccgtcttcc tccactccct
      121 cttccccttg ctctctgctg tgttgctgcc caaggatgct ctttccggag cacttccttc
      181 tcggcgctgc accacgtgat gtcctctgag cggatcctcc ccgtgtctgg gtcctctccg
      241 ggcatctctc ctccctcacc caaccccatg ccgtcttcac tcgctgggtt cccttttcct
      301 tctccttctg gggcctgtgc catctctcgt ttcttaggat ggccttctcc gacggatgtc
      361 tcccttgcgt cccgcctccc cttcttgtag gcctgcatca tcaccgtttt tctggacaac
      421 cccaaagtac cccgtctccc tggctttagc cacctctcca tcctcttgct ttctttgcct
      481 ggacaccccg ttctcctgtg gattcgggtc acctctcact cctttcattt gggcagctcc
      541 cctacccccc ttacctctct agtctgtgct agctcttcca gccccctgtc atggcatctt
      601 ccaggggtcc gagagctcag ctagtcttct tcctccaacc cgggccccta tgtccacttc
      661 aggacagcat gtttgctgcc tccagggatc ctgtgtcccc gagctgggac caccttatat
      721 tcccagggcc ggttaatgtg gctctggttc tgggtacttt tatctgtccc ctccacccca
      781 cagtggggca agcttctgac ctcttctctt cctcccacag ggcctcgaga gatctggcag
      841 cggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggccctag
      901 gctcgagggt accatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
      961 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     1021 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     1081 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     1141 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     1201 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     1261 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     1321 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     1381 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     1441 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     1501 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     1561 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     1621 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     1681 ctatcgcctt cttgacgagt tcttctgagt ttaaacccgc tgatcagcct cgactgtgcc
     1741 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1801 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1861 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     1921 caatagcagg catgctgggg atgcggtggg ctctatggac tagcggtggc ggcctcgaca
     1981 ttgattattg actagtgtca tggagaagac ccaccttgca gatgtcctca ctggggctgg
     2041 cagagccggc aacctgccca aggctgctca gtccattagg agccagtagc ctggaagatg
     2101 tctttacccc cagcatcagt tcaagtggag cagcacataa ctcttgccct ctgccttcca
     2161 agattctggt gctgagactt atggagtgtc ttggaggttg ccttctgccc cccaaccctg
     2221 ctcccagctg gccctcccag gcctgggttg ctggcctctg ctttatcagg attctcaaga
     2281 gggacagctg gtttatgttg catgactgtt ccctgcatat ctgctctggt tttaaatagc
     2341 ttatctgagc agctggagga ccacatgggc ttatatggcg tggggtacat gttcctgtag
     2401 ccttgtccct ggcacctgcc aaaatagcag ccaacacccc ccacccccac cgccatcccc
     2461 ctgccccacc cgtcccctgt cgcacattcc tccctccgca gggctggctc accaggcccc
     2521 agcccacatg cctgcttaaa gccctctcca tcctctgcct cacccagtcc ccgctgagac
     2581 tgagcagacg cctccaggat cgcggccgcc atgaccgagt acaagcccac ggtgcgcctc
     2641 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     2701 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     2761 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     2821 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     2881 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     2941 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     3001 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     3061 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     3121 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     3181 tggtgcatga cccgcaagcc cggtgcctga cctcgaggaa gggcgaattc actcctcagg
     3241 tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc
     3301 actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc
     3361 tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt
     3421 gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt
     3481 tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa ggttggctat
     3541 aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag
     3601 ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat
     3661 ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc
     3721 cagtcatagc tgtccctctt ctcttatgga gatccctcga cctgcagccc aagcttggat
     3781 ccctcgagtt aattaaatct agaagtcgac agtactaagc tttgacagaa aagccccatc
     3841 cttaggcctc ctccttccta gtctcctgat attgggtcta acccccacct cctgttaggc
     3901 agattcctta tctggtgaca cacccccatt tcctggagcc atctctctcc ttgccagaac
     3961 ctctaaggtt tgcttacgat ggagccagag aggatcctgg gagggagagc ttggcagggg
     4021 gtgggaggga agggggggat gcgtgacctg cccggttctc agtggccacc ctgcgctacc
     4081 ctctcccaga acctgagctg ctctgacgcg gctgtctggt gcgtttcact gatcctggtg
     4141 ctgcagcttc cttacacttc ccaagaggag aagcagtttg gaaaaacaaa atcagaataa
     4201 gttggtcctg agttctaact ttggctcttc acctttctag tccccaattt atattgttcc
     4261 tccgtgcgtc agttttacct gtgagataag gccagtagcc agccccgtcc tggcagggct
     4321 gtggtgagga ggggggtgtc cgtgtggaaa actccctttg tgagaatggt gcgtcctagg
     4381 tgttcaccag gtcgtggccg cctctactcc ctttctcttt ctccatcctt ctttccttaa
     4441 agagtcccca gtgctatctg ggacatattc ctccgcccag agcagggtcc cgcttcccta
     4501 aggccctgct ctgggcttct gggtttgagt ccttggcaag cccaggagag gcgctcaggc
     4561 ttccctgtcc cccttcctcg tccaccatct catgcccctg gctctcctgc cccttcccta
     4621 caggggttcc tggctctgct ctagcgatcg ccaattcgcc ctatagtgag tcgtattaca
     4681 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4741 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     4801 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     4861 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4921 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4981 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5041 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5101 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5161 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5221 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5281 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5341 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5401 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5461 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5521 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5581 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5641 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5701 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5761 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5821 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5881 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5941 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6001 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6061 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6121 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6181 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6241 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6301 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6361 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6421 ctgagcgtca aaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6481 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6541 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6601 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6661 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6721 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6781 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6841 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6901 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6961 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7021 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7081 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     7141 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     7201 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     7261 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     7321 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     7381 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     7441 cagctatgac catgattacg ccaagctcga aattaaccct cactaaaggg aacaaaagct
     7501 gtgctttctc tgacca
//
