LOCUS       sequence-457181-        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(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    402..1205
                     /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    1212..1237
                     /label=SA
                     /note="splice acceptor site"
     CDS             1261..1314
                     /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             1324..1923
                     /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(1324..1343)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1820..1840
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1955..1972)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1961..2185
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2873..3222
                     /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    2881..2899
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2917..2935
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2953..2971
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2989..3007
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3025..3043
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3061..3079
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3097..3115
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3161..3185
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3229..3238
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3235..3945
                     /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(3380..3398)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3648..3667
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4142..4223
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4179..4198)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    4249..5085
                     /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(5104..5120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5104..5120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5117..5139)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5128..5144
                     /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(5152..5158,5159..5176,5177..5182))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5197..5218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5335..5352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5506..6094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5586..5605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6265..7056,7057..7125))
                     /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     6888..6907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7126..7230)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7298..7316
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtgctttctc tgaccagcat
      421 tctctcccct gggcctgtgc cgctttctgt ctgtagcttg tggcctgggt cacctctacg
      481 gctggcccag atccttccct gccgcctcct tcaggttccg tcttcctcca ctccctcttc
      541 cccttgctct ctgctgtgtt gctgcccaag gatgctcttt ccggagcact tccttctcgg
      601 cgctgcacca cgtgatgtcc tctgagcgga tcctccccgt gtctgggtcc tctccgggca
      661 tctctcctcc ctcacccaac cccatgccgt cttcactcgc tgggttccct tttccttctc
      721 cttctggggc ctgtgccatc tctcgtttct taggatggcc ttctccgacg gatgtctccc
      781 ttgcgtcccg cctccccttc ttgtaggcct gcatcatcac cgtttttctg gacaacccca
      841 aagtaccccg tctccctggc tttagccacc tctccatcct cttgctttct ttgcctggac
      901 accccgttct cctgtggatt cgggtcacct ctcactcctt tcatttgggc agctccccta
      961 ccccccttac ctctctagtc tgtgctagct cttccagccc cctgtcatgg catcttccag
     1021 gggtccgaga gctcagctag tcttcttcct ccaacccggg cccctatgtc cacttcagga
     1081 cagcatgttt gctgcctcca gggatcctgt gtccccgagc tgggaccacc ttatattccc
     1141 agggccggtt aatgtggctc tggttctggg tacttttatc tgtcccctcc accccacagt
     1201 ggggcaagct tctgacctct tctcttcctc ccacagggcc tcgagagatc tggcagcgga
     1261 gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccctaggctc
     1321 gagatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     1381 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     1441 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     1501 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     1561 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     1621 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     1681 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     1741 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     1801 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     1861 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     1921 tgatctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     1981 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2041 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2101 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2161 gctggggatg cggtgggctc tatggtcact cgagtacggg aggtggtccc tgcagttacg
     2221 ccaatgataa cccccgccag aaaaatctta gtagccttcc ctttttgttt tccgtgcccc
     2281 aactcggcgg attgactcgg ccccttccgg aaacacccga atcaacttct agtcaaatta
     2341 ttgttcacgc cgcaatgacc cacccctggc ccgcgtctgt ggaactgacc cctggtgtac
     2401 aggagagttc gctgctgaaa gtggtcccaa aggggtacta gtttttaagc tcccaactcc
     2461 ccctccccca gcgtctggag gattccacac cctcgcaccg caggggcgag gaagtgggcg
     2521 gagtccggtt ttggcgccag ccgctgaggc tgccaagcag aaaagccacc gctgaggaga
     2581 ctccggtcac tgtcctcgcc ccgcctcccc cttccctccc cttggggacc accgggcgcc
     2641 acgccgcgaa cggtaagtgc cgcggtcgtc ggcgcctccg ccctccccct agggccccaa
     2701 ttcccagcgg gcgcggcgcg cggcccctcc ccccgccggg cgcgcgcccg ctgccccgcc
     2761 cttcgtggcc gcccggcgtg ggcggtgcca cccctccccc cggcggcccc gcgcgcagct
     2821 cccggctccc tcccccttcg gatgtggctt gagctgtagg cgcggaggac tcgagtttac
     2881 tccctatcag tgatagagaa cgtatgaaga gtttactccc tatcagtgat agagaacgta
     2941 tgcagacttt actccctatc agtgatagag aacgtataag gagtttactc cctatcagtg
     3001 atagagaacg tatgaccagt ttactcccta tcagtgatag agaacgtatc tacagtttac
     3061 tccctatcag tgatagagaa cgtatatcca gtttactccc tatcagtgat agagaacgta
     3121 tgtcgaggta ggcgtgtacg gtgggcgcct ataaaagcag agctcgttta gtgaaccgtc
     3181 agatcgcctg gagcaattcc acaacacttt tgtcttatac ttactagtgc caccatggtg
     3241 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     3301 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     3361 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     3421 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     3481 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     3541 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     3601 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     3661 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     3721 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     3781 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     3841 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     3901 gagggccgcc actccaccgg cggcatggac gagctgtaca agtaagaatt cgagctcggt
     3961 acccggggat cctctagtca gctgacgcgt gctagcgcgg ccgcatcgat aagcttgtcg
     4021 acgatatctc tagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt
     4081 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     4141 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4201 aaataaagca tttttttcac tgccttgaca gtactcttaa gtcgacttac tagggacagg
     4261 attggtgaca gaaaagcccc atccttaggc ctcctccttc ctagtctcct gatattgggt
     4321 ctaaccccca cctcctgtta ggcagattcc ttatctggtg acacaccccc atttcctgga
     4381 gccatctctc tccttgccag aacctctaag gtttgcttac gatggagcca gagaggatcc
     4441 tgggagggag agcttggcag ggggtgggag ggaagggggg gatgcgtgac ctgcccggtt
     4501 ctcagtggcc accctgcgct accctctccc agaacctgag ctgctctgac gcggctgtct
     4561 ggtgcgtttc actgatcctg gtgctgcagc ttccttacac ttcccaagag gagaagcagt
     4621 ttggaaaaac aaaatcagaa taagttggtc ctgagttcta actttggctc ttcacctttc
     4681 tagtccccaa tttatattgt tcctccgtgc gtcagtttta cctgtgagat aaggccagta
     4741 gccagccccg tcctggcagg gctgtggtga ggaggggggt gtccgtgtgg aaaactccct
     4801 ttgtgagaat ggtgcgtcct aggtgttcac caggtcgtgg ccgcctctac tccctttctc
     4861 tttctccatc cttctttcct taaagagtcc ccagtgctat ctgggacata ttcctccgcc
     4921 cagagcaggg tcccgcttcc ctaaggccct gctctgggct tctgggtttg agtccttggc
     4981 aagcccagga gaggcgctca ggcttccctg tcccccttcc tcgtccacca tctcatgccc
     5041 ctggctctcc tgccccttcc ctacaggggt tcctggctct gctctaagct tggcgtaatc
     5101 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5161 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5221 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5281 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5341 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5401 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5461 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5521 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5581 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5641 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5701 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5761 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5821 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5881 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5941 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6001 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6061 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6121 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6181 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6241 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6301 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6361 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6421 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6481 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6541 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6601 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6661 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6721 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6781 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6841 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6901 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6961 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7021 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7081 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7141 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7201 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7261 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     7321 tcgtc
//
