LOCUS       sequence-456997-        7325 bp    DNA     circular SYN 15-APR-2025
DEFINITION  It presents the UCOE sequence and the TRE3VG inducible promoter 
            allowing a more stable dox-dependent inducible activation of mCherry
            though the OPTi-OX platform across differentiation of hiPSCs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7325)
  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 7325)
  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_UCOE_TRE3VG_mCherry-Responder
FEATURES             Location/Qualifiers
     source          1..7325
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..41)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     141..163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(201..219)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        287..391
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(392..460,461..1252)
                     /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(610..629)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1423..2011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1912..1931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2165..2182
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2299..2320
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2335..2340,2341..2358,2359..2365)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2373..2389
                     /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     2378..2400
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2397..2413
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2397..2413
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    2432..3268
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     polyA_signal    3294..3375
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3319..3338
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3572..4282)
                     /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(3850..3869)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4119..4137
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      4279..4288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4295..4644)
                     /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"
     primer_bind     complement(4332..4356)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    4402..4420
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4438..4456
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4474..4492
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4510..4528
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4546..4564
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4582..4600
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4618..4636
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     polyA_signal    5332..5556
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5545..5562
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5594..6193)
                     /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(5677..5697)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     6174..6193
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(6203..6256)
                     /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"
     misc_feature    6280..6305
                     /label=SA
                     /note="splice acceptor site"
     misc_feature    6312..7115
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(7122..7139)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7122..7138)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7131..7153)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
       61 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
      121 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
      181 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
      241 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
      301 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
      361 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
      421 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
      481 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
      541 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
      601 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
      661 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
      721 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
      781 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
      841 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
      901 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
      961 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1021 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1081 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     1141 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     1201 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     1261 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     1321 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     1381 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1441 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1501 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     1561 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     1621 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     1681 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     1741 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     1801 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     1861 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     1921 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     1981 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2041 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     2101 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     2161 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     2221 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     2281 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     2341 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     2401 aaacagctat gaccatgatt acgccaagct tagagcagag ccaggaaccc ctgtagggaa
     2461 ggggcaggag agccaggggc atgagatggt ggacgaggaa gggggacagg gaagcctgag
     2521 cgcctctcct gggcttgcca aggactcaaa cccagaagcc cagagcaggg ccttagggaa
     2581 gcgggaccct gctctgggcg gaggaatatg tcccagatag cactggggac tctttaagga
     2641 aagaaggatg gagaaagaga aagggagtag aggcggccac gacctggtga acacctagga
     2701 cgcaccattc tcacaaaggg agttttccac acggacaccc ccctcctcac cacagccctg
     2761 ccaggacggg gctggctact ggccttatct cacaggtaaa actgacgcac ggaggaacaa
     2821 tataaattgg ggactagaaa ggtgaagagc caaagttaga actcaggacc aacttattct
     2881 gattttgttt ttccaaactg cttctcctct tgggaagtgt aaggaagctg cagcaccagg
     2941 atcagtgaaa cgcaccagac agccgcgtca gagcagctca ggttctggga gagggtagcg
     3001 cagggtggcc actgagaacc gggcaggtca cgcatccccc ccttccctcc caccccctgc
     3061 caagctctcc ctcccaggat cctctctggc tccatcgtaa gcaaacctta gaggttctgg
     3121 caaggagaga gatggctcca ggaaatgggg gtgtgtcacc agataaggaa tctgcctaac
     3181 aggaggtggg ggttagaccc aatatcagga gactaggaag gaggaggcct aaggatgggg
     3241 cttttctgtc accaatcctg tccctagtaa gtcgacttaa gagtactgtc aaggcagtga
     3301 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     3361 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     3421 gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat
     3481 cctctagaga tatcgtcgac aagcttatcg atgcggccgc gctagcacgc gtcagctgac
     3541 tagaggatcc ccgggtaccg agctcgaatt cttacttgta cagctcgtcc atgccgccgg
     3601 tggagtggcg gccctcggcg cgttcgtact gttccacgat ggtgtagtcc tcgttgtggg
     3661 aggtgatgtc caacttgatg ttgacgttgt aggcgccggg cagctgcacg ggcttcttgg
     3721 ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc gtccttcagc ttcagcctct
     3781 gcttgatctc gcccttcagg gcgccgtcct cggggtacat ccgctcggag gaggcctccc
     3841 agcccatggt cttcttctgc attacggggc cgtcggaggg gaagttggtg ccgcgcagct
     3901 tcaccttgta gatgaactcg ccgtcctgca gggaggagtc ctgggtcacg gtcaccacgc
     3961 cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag gacagcttca
     4021 agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac atgaactgag
     4081 gggacaggat gtcccaggcg aagggcaggg ggccaccctt ggtcaccttc agcttggcgg
     4141 tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac tcgtggccgt
     4201 tcacggagcc ctccatgtgc accttgaagc gcatgaactc cttgatgatg gccatgttat
     4261 cctcctcgcc cttgctcacc atggtggcac tagtaagtat aagacaaaag tgttgtggaa
     4321 ttgctccagg cgatctgacg gttcactaaa cgagctctgc ttttataggc gcccaccgta
     4381 cacgcctacc tcgacatacg ttctctatca ctgataggga gtaaactgga tatacgttct
     4441 ctatcactga tagggagtaa actgtagata cgttctctat cactgatagg gagtaaactg
     4501 gtcatacgtt ctctatcact gatagggagt aaactcctta tacgttctct atcactgata
     4561 gggagtaaag tctgcatacg ttctctatca ctgataggga gtaaactctt catacgttct
     4621 ctatcactga tagggagtaa actcgagtcc tccgcgccta cagctcaagc cacatccgaa
     4681 gggggaggga gccgggagct gcgcgcgggg ccgccggggg gaggggtggc accgcccacg
     4741 ccgggcggcc acgaagggcg gggcagcggg cgcgcgcccg gcggggggag gggccgcgcg
     4801 ccgcgcccgc tgggaattgg ggccctaggg ggagggcgga ggcgccgacg accgcggcac
     4861 ttaccgttcg cggcgtggcg cccggtggtc cccaagggga gggaaggggg aggcggggcg
     4921 aggacagtga ccggagtctc ctcagcggtg gcttttctgc ttggcagcct cagcggctgg
     4981 cgccaaaacc ggactccgcc cacttcctcg cccctgcggt gcgagggtgt ggaatcctcc
     5041 agacgctggg ggagggggag ttgggagctt aaaaactagt acccctttgg gaccactttc
     5101 agcagcgaac tctcctgtac accaggggtc agttccacag acgcgggcca ggggtgggtc
     5161 attgcggcgt gaacaataat ttgactagaa gttgattcgg gtgtttccgg aaggggccga
     5221 gtcaatccgc cgagttgggg cacggaaaac aaaaagggaa ggctactaag atttttctgg
     5281 cgggggttat cattggcgta actgcaggga ccacctcccg tactcgagtg accatagagc
     5341 ccaccgcatc cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc
     5401 ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat
     5461 tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg
     5521 ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag cgggtttaaa
     5581 cgggccctct agatcaggca ccgggcttgc gggtcatgca ccaggtgcgc ggtccttcgg
     5641 gcacctcgac gtcggcggtg acggtgaagc cgagccgctc gtagaagggg aggttgcggg
     5701 gcgcggaggt ctccaggaag gcgggcaccc cggcgcgctc ggccgcctcc actccgggga
     5761 gcacgacggc gctgcccaga cccttgccct ggtggtcggg cgagacgccg acggtggcca
     5821 ggaaccacgc gggctccttg ggccggtgcg gcgccaggag gccttccatc tgttgctgcg
     5881 cggccagccg ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg aacaccgccc
     5941 ccgcttcgac gctctccggc gtggtccaga ccgccaccgc ggcgccgtcg tccgcgaccc
     6001 acaccttgcc gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc tcggtgaccc
     6061 gctcgatgtg gcggtccgga tcgacggtgt ggcgcgtggc ggggtagtcg gcgaacgcgg
     6121 cggcgagggt gcgtacggcc ctggggacgt cgtcgcgggt ggcgaggcgc accgtgggct
     6181 tgtactcggt catctcgagc ctagggccgg gattctcctc cacgtcaccg catgttagaa
     6241 gacttcctct gccctctccg ctgccagatc tctcgaggcc ctgtgggagg aagagaagag
     6301 gtcagaagct tgccccactg tggggtggag gggacagata aaagtaccca gaaccagagc
     6361 cacattaacc ggccctggga atataaggtg gtcccagctc ggggacacag gatccctgga
     6421 ggcagcaaac atgctgtcct gaagtggaca taggggcccg ggttggagga agaagactag
     6481 ctgagctctc ggacccctgg aagatgccat gacagggggc tggaagagct agcacagact
     6541 agagaggtaa ggggggtagg ggagctgccc aaatgaaagg agtgagaggt gacccgaatc
     6601 cacaggagaa cggggtgtcc aggcaaagaa agcaagagga tggagaggtg gctaaagcca
     6661 gggagacggg gtactttggg gttgtccaga aaaacggtga tgatgcaggc ctacaagaag
     6721 gggaggcggg acgcaaggga gacatccgtc ggagaaggcc atcctaagaa acgagagatg
     6781 gcacaggccc cagaaggaga aggaaaaggg aacccagcga gtgaagacgg catggggttg
     6841 ggtgagggag gagagatgcc cggagaggac ccagacacgg ggaggatccg ctcagaggac
     6901 atcacgtggt gcagcgccga gaaggaagtg ctccggaaag agcatccttg ggcagcaaca
     6961 cagcagagag caaggggaag agggagtgga ggaagacgga acctgaagga ggcggcaggg
     7021 aaggatctgg gccagccgta gaggtgaccc aggccacaag ctacagacag aaagcggcac
     7081 aggcccaggg gagagaatgc tggtcagaga aagcacaatt cactggccgt cgttttacaa
     7141 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     7201 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     7261 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     7321 tcaca
//
