LOCUS       sequence-422164-        6907 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 6907)
  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 6907)
  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-Blast-TNNT2-Zeo
FEATURES             Location/Qualifiers
     source          1..6907
                     /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             914..1309
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(1344..1361)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1350..1574
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     regulatory      2221..2230
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2227..2601
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     primer_bind     complement(2699..2718)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2764..2819
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2818..2837)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     misc_feature    3375..3904
                     /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(4050..4069)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4051..4069)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4076..4093)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4076..4092)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4085..4107)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4234..4689
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4321..4340)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4531..4552
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4715..4819
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4820..4888,4889..5680)
                     /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(5038..5057)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5851..6439
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6340..6359
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6593..6610
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6727..6748
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6763..6768,6769..6786,6787..6793)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6801..6817
                     /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     6806..6828
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6825..6841
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6825..6841
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        6862..6880
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(6893..6907,1..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 accatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
      961 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
     1021 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
     1081 tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
     1141 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
     1201 gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
     1261 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggcc tgcagctgca
     1321 gtaggtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     1381 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1441 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1501 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     1561 ggtgggctct atggactagc ggtggcggcc tcgacattga ttattgacta gtgtcatgga
     1621 gaagacccac cttgcagatg tcctcactgg ggctggcaga gccggcaacc tgcccaaggc
     1681 tgctcagtcc attaggagcc agtagcctgg aagatgtctt tacccccagc atcagttcaa
     1741 gtggagcagc acataactct tgccctctgc cttccaagat tctggtgctg agacttatgg
     1801 agtgtcttgg aggttgcctt ctgcccccca accctgctcc cagctggccc tcccaggcct
     1861 gggttgctgg cctctgcttt atcaggattc tcaagaggga cagctggttt atgttgcatg
     1921 actgttccct gcatatctgc tctggtttta aatagcttat ctgagcagct ggaggaccac
     1981 atgggcttat atggcgtggg gtacatgttc ctgtagcctt gtccctggca cctgccaaaa
     2041 tagcagccaa caccccccac ccccaccgcc atccccctgc cccacccgtc ccctgtcgca
     2101 cattcctccc tccgcagggc tggctcacca ggccccagcc cacatgcctg cttaaagccc
     2161 tctccatcct ctgcctcacc cagtccccgc tgagactgag cagacgcctc caggatcgcg
     2221 gccgccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga
     2281 gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc
     2341 gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg
     2401 ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg
     2461 tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc
     2521 gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc
     2581 gtggccgagg agcaggactg acctcgagga agggcgaatt cactcctcag gtgcaggctg
     2641 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
     2701 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg
     2761 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac
     2821 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga
     2881 gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta taaagaggtc
     2941 atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa gccttgactt
     3001 gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca tccctaaaat
     3061 tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc ccagtcatag
     3121 ctgtccctct tctcttatgg agatccctcg acctgcagcc caagcttgga tccctcgagt
     3181 taattaaatc tagaagtcga cagtactaag ctttgacaga aaagccccat ccttaggcct
     3241 cctccttcct agtctcctga tattgggtct aacccccacc tcctgttagg cagattcctt
     3301 atctggtgac acacccccat ttcctggagc catctctctc cttgccagaa cctctaaggt
     3361 ttgcttacga tggagccaga gaggatcctg ggagggagag cttggcaggg ggtgggaggg
     3421 aaggggggga tgcgtgacct gcccggttct cagtggccac cctgcgctac cctctcccag
     3481 aacctgagct gctctgacgc ggctgtctgg tgcgtttcac tgatcctggt gctgcagctt
     3541 ccttacactt cccaagagga gaagcagttt ggaaaaacaa aatcagaata agttggtcct
     3601 gagttctaac tttggctctt cacctttcta gtccccaatt tatattgttc ctccgtgcgt
     3661 cagttttacc tgtgagataa ggccagtagc cagccccgtc ctggcagggc tgtggtgagg
     3721 aggggggtgt ccgtgtggaa aactcccttt gtgagaatgg tgcgtcctag gtgttcacca
     3781 ggtcgtggcc gcctctactc cctttctctt tctccatcct tctttcctta aagagtcccc
     3841 agtgctatct gggacatatt cctccgccca gagcagggtc ccgcttccct aaggccctgc
     3901 tctgggcttc tgggtttgag tccttggcaa gcccaggaga ggcgctcagg cttccctgtc
     3961 ccccttcctc gtccaccatc tcatgcccct ggctctcctg ccccttccct acaggggttc
     4021 ctggctctgc tctagcgatc gccaattcgc cctatagtga gtcgtattac aattcactgg
     4081 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4141 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     4201 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     4261 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     4321 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4381 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4441 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     4501 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     4561 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     4621 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     4681 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4741 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4801 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4861 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4921 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4981 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5041 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5101 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5161 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5221 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5281 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5341 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5401 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5461 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5521 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5581 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5641 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5701 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5761 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5821 aaaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5881 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5941 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6001 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6061 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6121 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6181 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6241 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6301 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6361 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6421 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6481 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     6541 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     6601 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     6661 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     6721 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     6781 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     6841 ccatgattac gccaagctcg aaattaaccc tcactaaagg gaacaaaagc tgtgctttct
     6901 ctgacca
//
