LOCUS       sequence-457157-        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"
     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        2192..2559
                     /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    2200..2218
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2236..2254
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2272..2290
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2308..2326
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2344..2362
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2380..2398
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2416..2434
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      2565..2574
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2571..2573,2574..2576,2577..3290)
                     /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(2571..2573,2574..2576,2577..3287)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2616..2637)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2877..2896)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3224..3245
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3487..3568
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3524..3543)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    3594..4430
                     /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(4449..4465)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4449..4465)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4462..4484)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4473..4489
                     /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(4497..4503,4504..4521,4522..4527))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4542..4563
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4680..4697)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4851..5439)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4931..4950)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5610..6401,6402..6470))
                     /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     6233..6252
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6471..6575)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6643..6661
                     /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 cgagtttact ccctatcagt gatagagaac
     2221 gtatgaagag tttactccct atcagtgata gagaacgtat gcagacttta ctccctatca
     2281 gtgatagaga acgtataagg agtttactcc ctatcagtga tagagaacgt atgaccagtt
     2341 tactccctat cagtgataga gaacgtatct acagtttact ccctatcagt gatagagaac
     2401 gtatatccag tttactccct atcagtgata gagaacgtat gtcgagtgct tatgtaaacc
     2461 agggcgccta taaaagagtg ctgatttttt gagtaaactt caattccaca acacttttgt
     2521 cttataccaa ctttccgtac cacttcctac cctcgtaaac tagtgccacc atggtgagca
     2581 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2641 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2701 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2761 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2821 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2881 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2941 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     3001 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     3061 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     3121 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     3181 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     3241 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcgagc
     3301 tcggtacccg gggatcctct agtcagctga cgcgtgctag cgcggccgca tcgataagct
     3361 tgtcgacgat atctctagag gatcataatc agccatacca catttgtaga ggttttactt
     3421 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     3481 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3541 ttcacaaata aagcattttt ttcactgcct tgacagtact cttaagtcga cttactaggg
     3601 acaggattgg tgacagaaaa gccccatcct taggcctcct ccttcctagt ctcctgatat
     3661 tgggtctaac ccccacctcc tgttaggcag attccttatc tggtgacaca cccccatttc
     3721 ctggagccat ctctctcctt gccagaacct ctaaggtttg cttacgatgg agccagagag
     3781 gatcctggga gggagagctt ggcagggggt gggagggaag ggggggatgc gtgacctgcc
     3841 cggttctcag tggccaccct gcgctaccct ctcccagaac ctgagctgct ctgacgcggc
     3901 tgtctggtgc gtttcactga tcctggtgct gcagcttcct tacacttccc aagaggagaa
     3961 gcagtttgga aaaacaaaat cagaataagt tggtcctgag ttctaacttt ggctcttcac
     4021 ctttctagtc cccaatttat attgttcctc cgtgcgtcag ttttacctgt gagataaggc
     4081 cagtagccag ccccgtcctg gcagggctgt ggtgaggagg ggggtgtccg tgtggaaaac
     4141 tccctttgtg agaatggtgc gtcctaggtg ttcaccaggt cgtggccgcc tctactccct
     4201 ttctctttct ccatccttct ttccttaaag agtccccagt gctatctggg acatattcct
     4261 ccgcccagag cagggtcccg cttccctaag gccctgctct gggcttctgg gtttgagtcc
     4321 ttggcaagcc caggagaggc gctcaggctt ccctgtcccc cttcctcgtc caccatctca
     4381 tgcccctggc tctcctgccc cttccctaca ggggttcctg gctctgctct aagcttggcg
     4441 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4501 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4561 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4621 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4681 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4741 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4801 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4861 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4921 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4981 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5041 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5101 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5161 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5221 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5281 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5341 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5401 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5461 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5521 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5581 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5641 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5701 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5761 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5821 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5881 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5941 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6001 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6061 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6121 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6181 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6241 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6301 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6361 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6421 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6481 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6541 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6601 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     6661 ccctttcgtc
//
