LOCUS       sequence-456992-        6670 bp    DNA     circular SYN 15-APR-2025
DEFINITION  It presents the T11 inducible promoter allowing the dox-dependent 
            inducible activation of EGFP through the OPTi-OX platform.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6670)
  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 6670)
  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_T11_EGFP-Responder
FEATURES             Location/Qualifiers
     source          1..6670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(61..80)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    131..967
                     /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(986..1002)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(986..1002)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(999..1021)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1010..1026
                     /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(1034..1040,1041..1058,1059..1064))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1079..1100
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1217..1234)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1388..1976)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1468..1487)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2147..2938,2939..3007))
                     /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     2770..2789
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3008..3112)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3180..3198
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3236..3258)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3358..3377
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3571..3593
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3585..3602
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3586..3602
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3609..4412
                     /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    4419..4444
                     /label=SA
                     /note="splice acceptor site"
     CDS             4468..4521
                     /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             4531..5130
                     /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(4531..4550)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5027..5047
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5162..5179)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5168..5392
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5399..5766
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    5407..5425
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5443..5461
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5479..5497
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5515..5533
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5551..5569
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5587..5605
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5623..5641
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      5772..5781
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5778..5780,5781..5783,5784..6497)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5778..5780,5781..5783,5784..6494)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5823..5844)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6084..6103)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6431..6452
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
       61 gcaatagcat cacaaatttc acaaataaag catttttttc actgccttga cagtactctt
      121 aagtcgactt actagggaca ggattggtga cagaaaagcc ccatccttag gcctcctcct
      181 tcctagtctc ctgatattgg gtctaacccc cacctcctgt taggcagatt ccttatctgg
      241 tgacacaccc ccatttcctg gagccatctc tctccttgcc agaacctcta aggtttgctt
      301 acgatggagc cagagaggat cctgggaggg agagcttggc agggggtggg agggaagggg
      361 gggatgcgtg acctgcccgg ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg
      421 agctgctctg acgcggctgt ctggtgcgtt tcactgatcc tggtgctgca gcttccttac
      481 acttcccaag aggagaagca gtttggaaaa acaaaatcag aataagttgg tcctgagttc
      541 taactttggc tcttcacctt tctagtcccc aatttatatt gttcctccgt gcgtcagttt
      601 tacctgtgag ataaggccag tagccagccc cgtcctggca gggctgtggt gaggaggggg
      661 gtgtccgtgt ggaaaactcc ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt
      721 ggccgcctct actccctttc tctttctcca tccttctttc cttaaagagt ccccagtgct
      781 atctgggaca tattcctccg cccagagcag ggtcccgctt ccctaaggcc ctgctctggg
      841 cttctgggtt tgagtccttg gcaagcccag gagaggcgct caggcttccc tgtccccctt
      901 cctcgtccac catctcatgc ccctggctct cctgcccctt ccctacaggg gttcctggct
      961 ctgctctaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     1021 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     1081 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1141 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1201 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     1261 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     1321 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1381 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1441 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1501 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1561 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1621 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     1681 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     1741 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     1801 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     1861 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     1921 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     1981 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2041 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2101 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2161 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2221 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2281 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2341 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2401 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2461 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2521 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2581 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     2641 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     2701 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     2761 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     2821 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     2881 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     2941 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3001 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3061 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     3121 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     3181 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     3241 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3301 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     3361 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
     3421 taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag
     3481 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     3541 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     3601 gtgaattgtg ctttctctga ccagcattct ctcccctggg cctgtgccgc tttctgtctg
     3661 tagcttgtgg cctgggtcac ctctacggct ggcccagatc cttccctgcc gcctccttca
     3721 ggttccgtct tcctccactc cctcttcccc ttgctctctg ctgtgttgct gcccaaggat
     3781 gctctttccg gagcacttcc ttctcggcgc tgcaccacgt gatgtcctct gagcggatcc
     3841 tccccgtgtc tgggtcctct ccgggcatct ctcctccctc acccaacccc atgccgtctt
     3901 cactcgctgg gttccctttt ccttctcctt ctggggcctg tgccatctct cgtttcttag
     3961 gatggccttc tccgacggat gtctcccttg cgtcccgcct ccccttcttg taggcctgca
     4021 tcatcaccgt ttttctggac aaccccaaag taccccgtct ccctggcttt agccacctct
     4081 ccatcctctt gctttctttg cctggacacc ccgttctcct gtggattcgg gtcacctctc
     4141 actcctttca tttgggcagc tcccctaccc cccttacctc tctagtctgt gctagctctt
     4201 ccagccccct gtcatggcat cttccagggg tccgagagct cagctagtct tcttcctcca
     4261 acccgggccc ctatgtccac ttcaggacag catgtttgct gcctccaggg atcctgtgtc
     4321 cccgagctgg gaccacctta tattcccagg gccggttaat gtggctctgg ttctgggtac
     4381 ttttatctgt cccctccacc ccacagtggg gcaagcttct gacctcttct cttcctccca
     4441 cagggcctcg agagatctgg cagcggagag ggcagaggaa gtcttctaac atgcggtgac
     4501 gtggaggaga atcccggccc taggctcgag atgaccgagt acaagcccac ggtgcgcctc
     4561 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     4621 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     4681 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     4741 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     4801 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     4861 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     4921 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     4981 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     5041 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     5101 tggtgcatga cccgcaagcc cggtgcctga tctagagggc ccgtttaaac ccgctgatca
     5161 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     5221 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     5281 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     5341 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggtcactcga
     5401 gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc agtgatagag
     5461 aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt ttactcccta
     5521 tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa cgtatctaca
     5581 gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc agtgatagag
     5641 aacgtatgtc gagtgcttat gtaaaccagg gcgcctataa aagagtgctg attttttgag
     5701 taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac ttcctaccct
     5761 cgtaaactag tgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     5821 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     5881 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     5941 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     6001 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     6061 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     6121 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     6181 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     6241 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     6301 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     6361 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     6421 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     6481 acgagctgta caagtaagaa ttcgagctcg gtacccgggg atcctctagt cagctgacgc
     6541 gtgctagcgc ggccgcatcg ataagcttgt cgacgatatc tctagaggat cataatcagc
     6601 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     6661 ctgaaacata
//
