LOCUS       sequence-456994-        6644 bp    DNA     circular SYN 15-APR-2025
DEFINITION  It presents the TRE3VG inducible promoter allowing the dox-dependent
            inducible activation of mCherry through the OPTi-OX platform.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6644)
  AUTHORS   Guichardaz M, Sveva B, Balmas E, Bertero A
  TITLE     Overcoming the Silencing of Doxycycline-Inducible Promoters in 
            hiPSC-derived Cardiomyocytes
  JOURNAL   Zenodo
REFERENCE   2  (bases 1 to 6644)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Zenodo"
COMMENT     Sequence Label: pAAV-puro_TRE3VG_mCherry-Responder
FEATURES             Location/Qualifiers
     source          1..6644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    208..289
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(245..264)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    315..1151
                     /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(1170..1186)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1170..1186)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1183..1205)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1194..1210
                     /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)."
     promoter        complement(join(1218..1224,1225..1242,1243..1248))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1263..1284
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1401..1418)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1572..2160)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1652..1671)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2331..3122,3123..3191))
                     /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     2954..2973
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3192..3296)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3364..3382
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3420..3442)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3542..3561
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3755..3777
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3769..3786
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3770..3786
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3793..4596
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    4603..4628
                     /label=SA
                     /note="splice acceptor site"
     CDS             4652..4705
                     /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             4715..5314
                     /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(4715..4734)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5211..5231
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5346..5363)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5352..5576
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5583..5932
                     /label=TRE3GV promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, optimized for 
                     retroviral and lentiviral vectors"
     protein_bind    5591..5609
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5627..5645
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5663..5681
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5699..5717
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5735..5753
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5771..5789
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5807..5825
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5871..5895
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      5939..5948
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5945..6644,1..11)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6090..6108)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6358..6377
                     /label=mCherry-F
                     /note="mCherry, forward primer"
ORIGIN
        1 tgtacaagta agaattcgag ctcggtaccc ggggatcctc tagtcagctg acgcgtgcta
       61 gcgcggccgc atcgataagc ttgtcgacga tatctctaga ggatcataat cagccatacc
      121 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
      181 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
      241 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgcc ttgacagtac
      301 tcttaagtcg acttactagg gacaggattg gtgacagaaa agccccatcc ttaggcctcc
      361 tccttcctag tctcctgata ttgggtctaa cccccacctc ctgttaggca gattccttat
      421 ctggtgacac acccccattt cctggagcca tctctctcct tgccagaacc tctaaggttt
      481 gcttacgatg gagccagaga ggatcctggg agggagagct tggcaggggg tgggagggaa
      541 gggggggatg cgtgacctgc ccggttctca gtggccaccc tgcgctaccc tctcccagaa
      601 cctgagctgc tctgacgcgg ctgtctggtg cgtttcactg atcctggtgc tgcagcttcc
      661 ttacacttcc caagaggaga agcagtttgg aaaaacaaaa tcagaataag ttggtcctga
      721 gttctaactt tggctcttca cctttctagt ccccaattta tattgttcct ccgtgcgtca
      781 gttttacctg tgagataagg ccagtagcca gccccgtcct ggcagggctg tggtgaggag
      841 gggggtgtcc gtgtggaaaa ctccctttgt gagaatggtg cgtcctaggt gttcaccagg
      901 tcgtggccgc ctctactccc tttctctttc tccatccttc tttccttaaa gagtccccag
      961 tgctatctgg gacatattcc tccgcccaga gcagggtccc gcttccctaa ggccctgctc
     1021 tgggcttctg ggtttgagtc cttggcaagc ccaggagagg cgctcaggct tccctgtccc
     1081 ccttcctcgt ccaccatctc atgcccctgg ctctcctgcc ccttccctac aggggttcct
     1141 ggctctgctc taagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     1201 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     1261 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1321 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1381 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1441 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     1501 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     1561 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     1621 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     1681 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     1741 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1801 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1861 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1921 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1981 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     2041 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2101 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2161 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2221 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2281 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2341 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2401 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2461 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2521 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2581 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     2641 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     2701 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     2761 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     2821 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     2881 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     2941 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3001 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3061 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3121 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3181 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     3241 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     3301 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     3361 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     3421 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     3481 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     3541 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     3601 tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg
     3661 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     3721 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     3781 gccagtgaat tgtgctttct ctgaccagca ttctctcccc tgggcctgtg ccgctttctg
     3841 tctgtagctt gtggcctggg tcacctctac ggctggccca gatccttccc tgccgcctcc
     3901 ttcaggttcc gtcttcctcc actccctctt ccccttgctc tctgctgtgt tgctgcccaa
     3961 ggatgctctt tccggagcac ttccttctcg gcgctgcacc acgtgatgtc ctctgagcgg
     4021 atcctccccg tgtctgggtc ctctccgggc atctctcctc cctcacccaa ccccatgccg
     4081 tcttcactcg ctgggttccc ttttccttct ccttctgggg cctgtgccat ctctcgtttc
     4141 ttaggatggc cttctccgac ggatgtctcc cttgcgtccc gcctcccctt cttgtaggcc
     4201 tgcatcatca ccgtttttct ggacaacccc aaagtacccc gtctccctgg ctttagccac
     4261 ctctccatcc tcttgctttc tttgcctgga caccccgttc tcctgtggat tcgggtcacc
     4321 tctcactcct ttcatttggg cagctcccct acccccctta cctctctagt ctgtgctagc
     4381 tcttccagcc ccctgtcatg gcatcttcca ggggtccgag agctcagcta gtcttcttcc
     4441 tccaacccgg gcccctatgt ccacttcagg acagcatgtt tgctgcctcc agggatcctg
     4501 tgtccccgag ctgggaccac cttatattcc cagggccggt taatgtggct ctggttctgg
     4561 gtacttttat ctgtcccctc caccccacag tggggcaagc ttctgacctc ttctcttcct
     4621 cccacagggc ctcgagagat ctggcagcgg agagggcaga ggaagtcttc taacatgcgg
     4681 tgacgtggag gagaatcccg gccctaggct cgagatgacc gagtacaagc ccacggtgcg
     4741 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     4801 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     4861 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     4921 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     4981 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     5041 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     5101 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     5161 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     5221 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     5281 cacctggtgc atgacccgca agcccggtgc ctgatctaga gggcccgttt aaacccgctg
     5341 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     5401 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     5461 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     5521 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggtcac
     5581 tcgagtttac tccctatcag tgatagagaa cgtatgaaga gtttactccc tatcagtgat
     5641 agagaacgta tgcagacttt actccctatc agtgatagag aacgtataag gagtttactc
     5701 cctatcagtg atagagaacg tatgaccagt ttactcccta tcagtgatag agaacgtatc
     5761 tacagtttac tccctatcag tgatagagaa cgtatatcca gtttactccc tatcagtgat
     5821 agagaacgta tgtcgaggta ggcgtgtacg gtgggcgcct ataaaagcag agctcgttta
     5881 gtgaaccgtc agatcgcctg gagcaattcc acaacacttt tgtcttatac ttactagtgc
     5941 caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     6001 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     6061 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     6121 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     6181 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     6241 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     6301 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     6361 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     6421 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     6481 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     6541 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     6601 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagc
//
