LOCUS       sequence-462054-        7734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline-regulatable EGFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7734)
  AUTHORS   Tominaga M, Shima Y, Nozaki K, Ito Y, Someda M, Shoya Y, Hashii N, 
            Obata C, Matsumoto-Kitano M, Suematsu K, Matsukawa T, Hosoya K, 
            Hashiba N, Kondo A, Ishii J
  TITLE     Designing strong inducible synthetic promoters in yeasts.
  JOURNAL   Nat Commun. 2024 Dec 19;15(1):10653. doi: 
            10.1038/s41467-024-54865-z.
  PUBMED    39702268
REFERENCE   2  (bases 1 to 7734)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-12-19"; volume: "15(1):10653. doi"; pages: " 
            10.1038/s41467-024-54865-z"
COMMENT     Sequence Label: pMT2013
FEATURES             Location/Qualifiers
     source          1..7734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(14..32)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        100..204
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(205..273,274..1065)
                     /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(423..442)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1236..1824
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1725..1744
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1978..1995
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2112..2133
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2148..2153,2154..2171,2172..2178)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2186..2202
                     /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     2191..2213
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2210..2226
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2210..2226
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    3960..3978
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3996..4014
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4031..4049
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4067..4085
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4103..4121
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4138..4156
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4174..4192
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4243..4261
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4279..4297
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4314..4332
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4350..4368
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4386..4404
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4421..4439
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4457..4475
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(4675..4677,4678..4680,4681..5391)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      5469..5715
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             6112..7134
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(7344..7361)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7344..7360)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7353..7375)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7569..7588)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7688..7710
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
       61 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
      121 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
      181 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
      241 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
      301 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
      361 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
      421 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
      481 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
      541 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
      601 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
      661 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
      721 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
      781 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
      841 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
      901 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
      961 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     1021 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     1081 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     1141 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     1201 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1261 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1321 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1381 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1441 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1501 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1561 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1621 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1681 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1741 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     1801 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     1861 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     1921 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     1981 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     2041 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     2101 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     2161 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     2221 tatgaccatg attacgccgc tagccatggt tgtgcgacag attcactgtg aaagactgtt
     2281 cattataccc acgtttcact gggagatgta agccttaggt gttttaccct gattagataa
     2341 tacaataacc aacagaaata cgagaatcta aactaatttc gatgattcat ttttcttttt
     2401 accgcgctgc ctcttttggc aattctttca cctatattct accttctctt tccttttgtt
     2461 ctaaacttat taccagctat ctatgtcgaa tcaagaagaa ggacttaaac tgtggggtgg
     2521 caggtttact ggggctactg accccttgat ggatttgtat aacgcttcct taccttacga
     2581 caagaaaatg tacaaggtgg atttagaagg aacaaaagtt tacactgagg gcctggagaa
     2641 aattaatttg ctaactaaag acgaactaag tgagattcat cgtggtctca aattgattga
     2701 agcagagtgg gcagaaggga agtttgttga gaagccaggg gatgaggata ttcacactgc
     2761 taatgaacgt cgcttgggtg agttgattgg tcgtggaatc tctggtaagg ttcataccgg
     2821 aaggtctaga aatgatcaag ttgccactga tatgcggttg tatgtcagag acaatctaac
     2881 tcagttggct gactatctga agcagttcat tcaagtaatc atcaagagag ctgaacagga
     2941 aatagacgtc ttgatgcccg gttatactca cttgcaaaga gctcaaccaa tcagatggtc
     3001 tcactggttg agcatgtatg ctacctattt cactgaagat tatgagagac tgaatcaaat
     3061 cgttaaaagg ttgaacaaat ccccattggg agctggagct ttggctggtc atccttatgg
     3121 aattgatcgt gaatacattg ctgagagatt agggtttgat tctgttattg gtaattcttt
     3181 ggccgctgtt tcagacagag attttgtagt cgaaaccatg ttctggtctt cgttgtttat
     3241 gaatcatatt tctcgattct cagaagattt gatcatttac tccactggag agtttggatt
     3301 tatcaagttg gcagatgctt attctactgg atcttctctg atgcctcaaa aaaaaaaccc
     3361 agactctttg gagttattga ggggtaaatc tggtagatgt tttggggcct tggctggttt
     3421 cctcatgtct attaagtcca ttccgtcaac ctataacaaa gatatgcaag aggataagga
     3481 gcctttattt gatactctaa tcactgtaga gcactcgatt ttgatagcat ccggtgtagt
     3541 ttctaccttg aacattgatg ccgaacgaat gaagaatgct ctaactatgg atatgctggc
     3601 tacagatctt gccgactatt tagttagaag gggagttcca ttcagagaaa ctcaccacat
     3661 ttctggtgaa tgtgtcagac aagccgagga gttgaacctt tctggtattg atcagttgtc
     3721 cctcgaacaa ttgaaatcca ttgactcccg ttttgaggct gatgtggctt caacgtttga
     3781 ctttgaagcc agtgttgaaa aaagaactgc caccggagga acttctaaga ctgctgtttt
     3841 aaagcaattg gatgcactga atgaaaagct agagtcttga ctgcaggtcg acctattacg
     3901 ccagcggatc ccgactcact atagggcgaa ttgggtaccg ggccccccct cgagtttact
     3961 ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgatg
     4021 tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat
     4081 agagaacgta tgtcgagttt actccctatc agtgatagag aacgatgtcg agtttactcc
     4141 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt
     4201 ggatgataat gcgattagtt ttttagcccc cctcgagttt actccctatc agtgatagag
     4261 aacgtatgtc gagtttactc cctatcagtg atagagaacg atgtcgagtt tactccctat
     4321 cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag
     4381 tttactccct atcagtgata gagaacgtat gtcgagttta tccctatcag tgatagagaa
     4441 cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtggatgat aatgctatcg
     4501 atagcaatat ataaacagaa ggaagctgcc ctgtcttaaa cctttttttt tatcatcatt
     4561 attagcttac tttcataatt gcgactggtt ccaattgaca agcttttgat tttaacgact
     4621 tttaacgaca acttgagaag atcaaaaaac aactaattat tcgaaacgac tagtatggtt
     4681 tctaagggtg aagagttgtt cactggtgtt gttccaatct tggttgagtt ggacggtgac
     4741 gttaacggac acaagttctc tgtttctggt gaaggtgagg gtgacgctac ttacggaaag
     4801 ttgactttga agttcatctg tactactggt aagttgccag ttccatggcc aactttggtt
     4861 actactttga cttacggtgt tcagtgtttc tccagatacc cagaccacat gaagcagcac
     4921 gatttcttca agtctgctat gccagagggt tacgttcaag agagaactat cttcttcaag
     4981 gacgacggta actacaagac tagagctgag gttaagttcg agggtgacac attggttaac
     5041 agaatcgagt tgaagggtat cgacttcaaa gaggacggaa acatcttggg tcacaagttg
     5101 gagtacaact acaactccca caacgtttac atcatggctg acaagcagaa gaacggtatc
     5161 aaggttaact tcaagatcag acacaacatc gaggacggtt ccgttcaatt ggctgaccac
     5221 taccaacaga acactccaat tggtgacggt ccagttttgt tgccagacaa ccactacttg
     5281 tccactcaat ccgctttgtc caaggaccca aacgagaaga gagatcacat ggttttgttg
     5341 gagttcgtta ctgctgctgg tatcactttg ggtatggacg agttgtacaa gtaactcgag
     5401 ccttagacat gactgttcct cagttcaagt tgggcactta cgagaagacc ggtcttgcta
     5461 gattctaatc aagaggatgt cagaatgcca tttgcctgag agatgcaggc ttcatttttg
     5521 atactttttt atttgtaacc tatatagtat aggatttttt ttgtcatttt gtttcttctc
     5581 gtacgagctt gctcctgatc agcctatctc gcagctgatg aatatcttgt ggtaggggtt
     5641 tgggaaaatc attcgagttt gatgtttttc ttggtatttc ccactcctct tcagagtaca
     5701 gaagattaag tgagacgttc gtttgtgcaa gcttagatcc ccgggtaccg agctcgaatt
     5761 cgccgtacca cttcaaaaca cccaagcaca gcatactaaa tttcccctct ttcttcctct
     5821 agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt
     5881 ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaaatt
     5941 tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg
     6001 tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc
     6061 ttgctcatta gaaagaaagc atagcaatct aatctaagtt ttaattacaa aatgaagaag
     6121 ccagagttga ctgctacttc cgttgagaag ttcttgatcg agaagttcga ctccgtttcc
     6181 gacttgatgc aattgtctga aggtgaagag tccagagctt tctcctttga cgttggtggt
     6241 agaggttacg ttttgagagt taattcctgt gctgacggtt tctacaagga cagatacgtt
     6301 tacagacact tcgcttccgc tgctttgcca attccagagg ttttggacat tggtgagttc
     6361 tccgagtcct tgacttactg tatctccaga agagctcagg gagttacatt gcaagacttg
     6421 ccagaaactg agttgccagc tgttttgcaa ccagttgctg aagctatgga cgctattgct
     6481 gctgctgact tgtctcaaac ttctggtttt ggtccattcg gtccacaggg tattggtcag
     6541 tacactactt ggagagactt catctgtgct atcgctgacc cacatgttta ccactggcaa
     6601 actgttatgg acgatactgt ttccgcttcc gttgctcagg ctttggacga attgatgttg
     6661 tgggctgaag attgtcctga ggttagacat ttggttcacg ctgacttcgg ttccaacaac
     6721 gttttgactg acaacggtag aatcactgct gttatcgact ggtccgaagc tatgttcggt
     6781 gactctcaat acgaggttgc taacatcttc ttttggagac catggttggc ttgtatggaa
     6841 cagcagacta gatacttcga gagaagacac ccagaattgg ctggttcccc aagattgaga
     6901 gcttacatgt tgagaatcgg tttggaccag ttgtaccagt ccttggttga tggtaacttc
     6961 gatgatgctg cttgggctca aggtagatgt gacgctatcg ttagatccgg tgctggtact
     7021 gttggtagaa ctcagattgc tagaagatcc gctgctgttt ggactgacgg ttgtgttgaa
     7081 gttttggctg actccggtaa cagaagacca tccactagac caagagctaa agaggttggt
     7141 agagtttaag tattaagaac gttatttata tttcaaattt ttcttttttt tctctacaga
     7201 cgctgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga
     7261 agatccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     7321 gctcttccgc ttcctcggaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     7381 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     7441 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     7501 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc
     7561 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     7621 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     7681 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgc
//
