LOCUS       sequence-458294-        7308 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 7308)
  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 7308)
  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: pMT2080
FEATURES             Location/Qualifiers
     source          1..7308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    3956..3974
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3991..4009
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4027..4045
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(4245..4247,4248..4250,4251..4961)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      5039..5285
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             5682..6704
                     /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(6914..6931)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6914..6930)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6923..6945)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7139..7158)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7258..7280
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccgctagc catggttgtg cgacagattc actgtgaaag actgttcatt
     2281 atacccacgt ttcactggga gatgtaagcc ttaggtgttt taccctgatt agataataca
     2341 ataaccaaca gaaatacgag aatctaaact aatttcgatg attcattttt ctttttaccg
     2401 cgctgcctct tttggcaatt ctttcaccta tattctacct tctctttcct tttgttctaa
     2461 acttattacc agctatctat gtcgaatcaa gaagaaggac ttaaactgtg gggtggcagg
     2521 tttactgggg ctactgaccc cttgatggat ttgtataacg cttccttacc ttacgacaag
     2581 aaaatgtaca aggtggattt agaaggaaca aaagtttaca ctgagggcct ggagaaaatt
     2641 aatttgctaa ctaaagacga actaagtgag attcatcgtg gtctcaaatt gattgaagca
     2701 gagtgggcag aagggaagtt tgttgagaag ccaggggatg aggatattca cactgctaat
     2761 gaacgtcgct tgggtgagtt gattggtcgt ggaatctctg gtaaggttca taccggaagg
     2821 tctagaaatg atcaagttgc cactgatatg cggttgtatg tcagagacaa tctaactcag
     2881 ttggctgact atctgaagca gttcattcaa gtaatcatca agagagctga acaggaaata
     2941 gacgtcttga tgcccggtta tactcacttg caaagagctc aaccaatcag atggtctcac
     3001 tggttgagca tgtatgctac ctatttcact gaagattatg agagactgaa tcaaatcgtt
     3061 aaaaggttga acaaatcccc attgggagct ggagctttgg ctggtcatcc ttatggaatt
     3121 gatcgtgaat acattgctga gagattaggg tttgattctg ttattggtaa ttctttggcc
     3181 gctgtttcag acagagattt tgtagtcgaa accatgttct ggtcttcgtt gtttatgaat
     3241 catatttctc gattctcaga agatttgatc atttactcca ctggagagtt tggatttatc
     3301 aagttggcag atgcttattc tactggatct tctctgatgc ctcaaaaaaa aaacccagac
     3361 tctttggagt tattgagggg taaatctggt agatgttttg gggccttggc tggtttcctc
     3421 atgtctatta agtccattcc gtcaacctat aacaaagata tgcaagagga taaggagcct
     3481 ttatttgata ctctaatcac tgtagagcac tcgattttga tagcatccgg tgtagtttct
     3541 accttgaaca ttgatgccga acgaatgaag aatgctctaa ctatggatat gctggctaca
     3601 gatcttgccg actatttagt tagaagggga gttccattca gagaaactca ccacatttct
     3661 ggtgaatgtg tcagacaagc cgaggagttg aacctttctg gtattgatca gttgtccctc
     3721 gaacaattga aatccattga ctcccgtttt gaggctgatg tggcttcaac gtttgacttt
     3781 gaagccagtg ttgaaaaaag aactgccacc ggaggaactt ctaagactgc tgttttaaag
     3841 caattggatg cactgaatga aaagctagag tcttgactgc aggtcgacct attacgccag
     3901 cggatcccga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag tttactccct
     3961 atcagtgata gagaacgtat gtcgagttta tccctatcag tgatagagaa cgtatgtcga
     4021 gtttactccc tatcagtgat agagaacgta tgtggatgat aatgctatcg atagcaatat
     4081 ataaacagaa ggaagctgcc ctgtcttaaa cctttttttt tatcatcatt attagcttac
     4141 tttcataatt gcgactggtt ccaattgaca agcttttgat tttaacgact tttaacgaca
     4201 acttgagaag atcaaaaaac aactaattat tcgaaacgac tagtatggtt tctaagggtg
     4261 aagagttgtt cactggtgtt gttccaatct tggttgagtt ggacggtgac gttaacggac
     4321 acaagttctc tgtttctggt gaaggtgagg gtgacgctac ttacggaaag ttgactttga
     4381 agttcatctg tactactggt aagttgccag ttccatggcc aactttggtt actactttga
     4441 cttacggtgt tcagtgtttc tccagatacc cagaccacat gaagcagcac gatttcttca
     4501 agtctgctat gccagagggt tacgttcaag agagaactat cttcttcaag gacgacggta
     4561 actacaagac tagagctgag gttaagttcg agggtgacac attggttaac agaatcgagt
     4621 tgaagggtat cgacttcaaa gaggacggaa acatcttggg tcacaagttg gagtacaact
     4681 acaactccca caacgtttac atcatggctg acaagcagaa gaacggtatc aaggttaact
     4741 tcaagatcag acacaacatc gaggacggtt ccgttcaatt ggctgaccac taccaacaga
     4801 acactccaat tggtgacggt ccagttttgt tgccagacaa ccactacttg tccactcaat
     4861 ccgctttgtc caaggaccca aacgagaaga gagatcacat ggttttgttg gagttcgtta
     4921 ctgctgctgg tatcactttg ggtatggacg agttgtacaa gtaactcgag ccttagacat
     4981 gactgttcct cagttcaagt tgggcactta cgagaagacc ggtcttgcta gattctaatc
     5041 aagaggatgt cagaatgcca tttgcctgag agatgcaggc ttcatttttg atactttttt
     5101 atttgtaacc tatatagtat aggatttttt ttgtcatttt gtttcttctc gtacgagctt
     5161 gctcctgatc agcctatctc gcagctgatg aatatcttgt ggtaggggtt tgggaaaatc
     5221 attcgagttt gatgtttttc ttggtatttc ccactcctct tcagagtaca gaagattaag
     5281 tgagacgttc gtttgtgcaa gcttagatcc ccgggtaccg agctcgaatt cgccgtacca
     5341 cttcaaaaca cccaagcaca gcatactaaa tttcccctct ttcttcctct agggtgtcgt
     5401 taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc
     5461 gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaaatt tttttttttg
     5521 atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg tcttcaattt
     5581 ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc ttgctcatta
     5641 gaaagaaagc atagcaatct aatctaagtt ttaattacaa aatgaagaag ccagagttga
     5701 ctgctacttc cgttgagaag ttcttgatcg agaagttcga ctccgtttcc gacttgatgc
     5761 aattgtctga aggtgaagag tccagagctt tctcctttga cgttggtggt agaggttacg
     5821 ttttgagagt taattcctgt gctgacggtt tctacaagga cagatacgtt tacagacact
     5881 tcgcttccgc tgctttgcca attccagagg ttttggacat tggtgagttc tccgagtcct
     5941 tgacttactg tatctccaga agagctcagg gagttacatt gcaagacttg ccagaaactg
     6001 agttgccagc tgttttgcaa ccagttgctg aagctatgga cgctattgct gctgctgact
     6061 tgtctcaaac ttctggtttt ggtccattcg gtccacaggg tattggtcag tacactactt
     6121 ggagagactt catctgtgct atcgctgacc cacatgttta ccactggcaa actgttatgg
     6181 acgatactgt ttccgcttcc gttgctcagg ctttggacga attgatgttg tgggctgaag
     6241 attgtcctga ggttagacat ttggttcacg ctgacttcgg ttccaacaac gttttgactg
     6301 acaacggtag aatcactgct gttatcgact ggtccgaagc tatgttcggt gactctcaat
     6361 acgaggttgc taacatcttc ttttggagac catggttggc ttgtatggaa cagcagacta
     6421 gatacttcga gagaagacac ccagaattgg ctggttcccc aagattgaga gcttacatgt
     6481 tgagaatcgg tttggaccag ttgtaccagt ccttggttga tggtaacttc gatgatgctg
     6541 cttgggctca aggtagatgt gacgctatcg ttagatccgg tgctggtact gttggtagaa
     6601 ctcagattgc tagaagatcc gctgctgttt ggactgacgg ttgtgttgaa gttttggctg
     6661 actccggtaa cagaagacca tccactagac caagagctaa agaggttggt agagtttaag
     6721 tattaagaac gttatttata tttcaaattt ttcttttttt tctctacaga cgctgtacgc
     6781 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga agatccagct
     6841 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     6901 ttcctcggaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     6961 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     7021 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc
     7081 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta
     7141 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     7201 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     7261 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcga
//
