LOCUS       sequence-456995-        6653 bp    DNA     circular SYN 15-APR-2025
DEFINITION  It presents the TRE3VG 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 6653)
  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 6653)
  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_EGFP-Responder
FEATURES             Location/Qualifiers
     source          1..6653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    16..97
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(53..72)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    123..959
                     /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(978..994)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(978..994)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(991..1013)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1002..1018
                     /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(1026..1032,1033..1050,1051..1056))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1071..1092
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1209..1226)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1380..1968)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1460..1479)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2139..2930,2931..2999))
                     /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     2762..2781
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3000..3104)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3172..3190
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3228..3250)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3350..3369
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3563..3585
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3577..3594
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3578..3594
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3601..4404
                     /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    4411..4436
                     /label=SA
                     /note="splice acceptor site"
     CDS             4460..4513
                     /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             4523..5122
                     /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(4523..4542)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5019..5039
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5154..5171)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5160..5384
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5391..5740
                     /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    5399..5417
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5435..5453
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5471..5489
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5507..5525
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5543..5561
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5579..5597
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5615..5633
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5679..5703
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      5747..5756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5753..5755,5756..5758,5759..6472)
                     /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(5753..5755,5756..5758,5759..6469)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5798..5819)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6059..6078)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6406..6427
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
       61 atcacaaatt tcacaaataa agcatttttt tcactgcctt gacagtactc ttaagtcgac
      121 ttactaggga caggattggt gacagaaaag ccccatcctt aggcctcctc cttcctagtc
      181 tcctgatatt gggtctaacc cccacctcct gttaggcaga ttccttatct ggtgacacac
      241 ccccatttcc tggagccatc tctctccttg ccagaacctc taaggtttgc ttacgatgga
      301 gccagagagg atcctgggag ggagagcttg gcagggggtg ggagggaagg gggggatgcg
      361 tgacctgccc ggttctcagt ggccaccctg cgctaccctc tcccagaacc tgagctgctc
      421 tgacgcggct gtctggtgcg tttcactgat cctggtgctg cagcttcctt acacttccca
      481 agaggagaag cagtttggaa aaacaaaatc agaataagtt ggtcctgagt tctaactttg
      541 gctcttcacc tttctagtcc ccaatttata ttgttcctcc gtgcgtcagt tttacctgtg
      601 agataaggcc agtagccagc cccgtcctgg cagggctgtg gtgaggaggg gggtgtccgt
      661 gtggaaaact ccctttgtga gaatggtgcg tcctaggtgt tcaccaggtc gtggccgcct
      721 ctactccctt tctctttctc catccttctt tccttaaaga gtccccagtg ctatctggga
      781 catattcctc cgcccagagc agggtcccgc ttccctaagg ccctgctctg ggcttctggg
      841 tttgagtcct tggcaagccc aggagaggcg ctcaggcttc cctgtccccc ttcctcgtcc
      901 accatctcat gcccctggct ctcctgcccc ttccctacag gggttcctgg ctctgctcta
      961 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     1021 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     1081 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     1141 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     1201 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     1261 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     1321 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     1381 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     1441 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     1501 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     1561 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     1621 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1681 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1741 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1801 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     1861 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1921 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1981 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     2041 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     2101 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     2161 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     2221 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     2281 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     2341 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     2401 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     2461 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     2521 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     2581 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     2641 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     2701 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     2761 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     2821 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     2881 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     2941 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3001 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     3061 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     3121 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     3181 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     3241 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     3301 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     3361 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     3421 aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     3481 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     3541 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg
     3601 tgctttctct gaccagcatt ctctcccctg ggcctgtgcc gctttctgtc tgtagcttgt
     3661 ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt
     3721 cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc
     3781 cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg
     3841 tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct
     3901 gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct
     3961 tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc
     4021 gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc
     4081 ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt
     4141 catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc
     4201 ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc
     4261 ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct
     4321 gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct
     4381 gtcccctcca ccccacagtg gggcaagctt ctgacctctt ctcttcctcc cacagggcct
     4441 cgagagatct ggcagcggag agggcagagg aagtcttcta acatgcggtg acgtggagga
     4501 gaatcccggc cctaggctcg agatgaccga gtacaagccc acggtgcgcc tcgccacccg
     4561 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     4621 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     4681 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     4741 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     4801 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     4861 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     4921 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     4981 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     5041 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     5101 gacccgcaag cccggtgcct gatctagagg gcccgtttaa acccgctgat cagcctcgac
     5161 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     5221 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     5281 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     5341 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggtcactc gagtttactc
     5401 cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag agaacgtatg
     5461 cagactttac tccctatcag tgatagagaa cgtataagga gtttactccc tatcagtgat
     5521 agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta cagtttactc
     5581 cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag agaacgtatg
     5641 tcgaggtagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt gaaccgtcag
     5701 atcgcctgga gcaattccac aacacttttg tcttatactt actagtgcca ccatggtgag
     5761 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     5821 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     5881 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     5941 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     6001 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     6061 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     6121 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     6181 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     6241 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     6301 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     6361 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     6421 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aagaattcga
     6481 gctcggtacc cggggatcct ctagtcagct gacgcgtgct agcgcggccg catcgataag
     6541 cttgtcgacg atatctctag aggatcataa tcagccatac cacatttgta gaggttttac
     6601 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aat
//
