LOCUS       sequence-457121-        7380 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 7380)
  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 7380)
  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: pMT2081
FEATURES             Location/Qualifiers
     source          1..7380
                     /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    3992..4010
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4028..4046
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4063..4081
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4099..4117
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(4317..4319,4320..4322,4323..5033)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      5111..5357
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             5754..6776
                     /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(6986..7003)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6986..7002)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6995..7017)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7211..7230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7330..7352
                     /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 ctccctatca gtgatagaga acgtatgtcg
     4021 agtttactcc ctatcagtga tagagaacgt atgtcgagtt tatccctatc agtgatagag
     4081 aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtggatg ataatgctat
     4141 cgatagcaat atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca
     4201 ttattagctt actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga
     4261 cttttaacga caacttgaga agatcaaaaa acaactaatt attcgaaacg actagtatgg
     4321 tttctaaggg tgaagagttg ttcactggtg ttgttccaat cttggttgag ttggacggtg
     4381 acgttaacgg acacaagttc tctgtttctg gtgaaggtga gggtgacgct acttacggaa
     4441 agttgacttt gaagttcatc tgtactactg gtaagttgcc agttccatgg ccaactttgg
     4501 ttactacttt gacttacggt gttcagtgtt tctccagata cccagaccac atgaagcagc
     4561 acgatttctt caagtctgct atgccagagg gttacgttca agagagaact atcttcttca
     4621 aggacgacgg taactacaag actagagctg aggttaagtt cgagggtgac acattggtta
     4681 acagaatcga gttgaagggt atcgacttca aagaggacgg aaacatcttg ggtcacaagt
     4741 tggagtacaa ctacaactcc cacaacgttt acatcatggc tgacaagcag aagaacggta
     4801 tcaaggttaa cttcaagatc agacacaaca tcgaggacgg ttccgttcaa ttggctgacc
     4861 actaccaaca gaacactcca attggtgacg gtccagtttt gttgccagac aaccactact
     4921 tgtccactca atccgctttg tccaaggacc caaacgagaa gagagatcac atggttttgt
     4981 tggagttcgt tactgctgct ggtatcactt tgggtatgga cgagttgtac aagtaactcg
     5041 agccttagac atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc
     5101 tagattctaa tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt
     5161 tgatactttt ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc
     5221 tcgtacgagc ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg
     5281 tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta
     5341 cagaagatta agtgagacgt tcgtttgtgc aagcttagat ccccgggtac cgagctcgaa
     5401 ttcgccgtac cacttcaaaa cacccaagca cagcatacta aatttcccct ctttcttcct
     5461 ctagggtgtc gttaattacc cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt
     5521 ttctttttct tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaaa
     5581 tttttttttt tgattttttt ctctttcgat gacctcccat tgatatttaa gttaataaac
     5641 ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt ctattacaac tttttttact
     5701 tcttgctcat tagaaagaaa gcatagcaat ctaatctaag ttttaattac aaaatgaaga
     5761 agccagagtt gactgctact tccgttgaga agttcttgat cgagaagttc gactccgttt
     5821 ccgacttgat gcaattgtct gaaggtgaag agtccagagc tttctccttt gacgttggtg
     5881 gtagaggtta cgttttgaga gttaattcct gtgctgacgg tttctacaag gacagatacg
     5941 tttacagaca cttcgcttcc gctgctttgc caattccaga ggttttggac attggtgagt
     6001 tctccgagtc cttgacttac tgtatctcca gaagagctca gggagttaca ttgcaagact
     6061 tgccagaaac tgagttgcca gctgttttgc aaccagttgc tgaagctatg gacgctattg
     6121 ctgctgctga cttgtctcaa acttctggtt ttggtccatt cggtccacag ggtattggtc
     6181 agtacactac ttggagagac ttcatctgtg ctatcgctga cccacatgtt taccactggc
     6241 aaactgttat ggacgatact gtttccgctt ccgttgctca ggctttggac gaattgatgt
     6301 tgtgggctga agattgtcct gaggttagac atttggttca cgctgacttc ggttccaaca
     6361 acgttttgac tgacaacggt agaatcactg ctgttatcga ctggtccgaa gctatgttcg
     6421 gtgactctca atacgaggtt gctaacatct tcttttggag accatggttg gcttgtatgg
     6481 aacagcagac tagatacttc gagagaagac acccagaatt ggctggttcc ccaagattga
     6541 gagcttacat gttgagaatc ggtttggacc agttgtacca gtccttggtt gatggtaact
     6601 tcgatgatgc tgcttgggct caaggtagat gtgacgctat cgttagatcc ggtgctggta
     6661 ctgttggtag aactcagatt gctagaagat ccgctgctgt ttggactgac ggttgtgttg
     6721 aagttttggc tgactccggt aacagaagac catccactag accaagagct aaagaggttg
     6781 gtagagttta agtattaaga acgttattta tatttcaaat ttttcttttt tttctctaca
     6841 gacgctgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc
     6901 gaagatccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6961 gcgctcttcc gcttcctcgg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     7021 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     7081 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     7141 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     7201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     7261 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     7321 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
//
