LOCUS       sequence-458298-        7063 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2,4-Diacetylphloroglucinol (DAPG)-regulatable EGFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7063)
  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 7063)
  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: pMT1475
FEATURES             Location/Qualifiers
     source          1..7063
                     /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"
     CDS             join(4000..4002,4003..4005,4006..4716)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      4794..5040
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             5437..6459
                     /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(6669..6686)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6669..6685)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6678..6700)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6894..6913)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7013..7035
                     /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 aggtcgactc tagaggatcc
     3901 tatgtatgat acgaaacgta ccgtatcgtt aaggtagcgt cagcaatata taaacagaag
     3961 gaagctgccc tgtcttaaac cttttttttt atcactagta tggtttctaa gggtgaagag
     4021 ttgttcactg gtgttgttcc aatcttggtt gagttggacg gtgacgttaa cggacacaag
     4081 ttctctgttt ctggtgaagg tgagggtgac gctacttacg gaaagttgac tttgaagttc
     4141 atctgtacta ctggtaagtt gccagttcca tggccaactt tggttactac tttgacttac
     4201 ggtgttcagt gtttctccag atacccagac cacatgaagc agcacgattt cttcaagtct
     4261 gctatgccag agggttacgt tcaagagaga actatcttct tcaaggacga cggtaactac
     4321 aagactagag ctgaggttaa gttcgagggt gacacattgg ttaacagaat cgagttgaag
     4381 ggtatcgact tcaaagagga cggaaacatc ttgggtcaca agttggagta caactacaac
     4441 tcccacaacg tttacatcat ggctgacaag cagaagaacg gtatcaaggt taacttcaag
     4501 atcagacaca acatcgagga cggttccgtt caattggctg accactacca acagaacact
     4561 ccaattggtg acggtccagt tttgttgcca gacaaccact acttgtccac tcaatccgct
     4621 ttgtccaagg acccaaacga gaagagagat cacatggttt tgttggagtt cgttactgct
     4681 gctggtatca ctttgggtat ggacgagttg tacaagtaac tcgagcctta gacatgactg
     4741 ttcctcagtt caagttgggc acttacgaga agaccggtct tgctagattc taatcaagag
     4801 gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg
     4861 taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc
     4921 tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg
     4981 agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga
     5041 cgttcgtttg tgcaagctta gatccccggg taccgagctc gaattcgccg taccacttca
     5101 aaacacccaa gcacagcata ctaaatttcc cctctttctt cctctagggt gtcgttaatt
     5161 acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga
     5221 aaaaggcaat aaaaattttt atcacgtttc tttttcttga aaattttttt ttttgatttt
     5281 tttctctttc gatgacctcc cattgatatt taagttaata aacggtcttc aatttctcaa
     5341 gtttcagttt catttttctt gttctattac aacttttttt acttcttgct cattagaaag
     5401 aaagcatagc aatctaatct aagttttaat tacaaaatga agaagccaga gttgactgct
     5461 acttccgttg agaagttctt gatcgagaag ttcgactccg tttccgactt gatgcaattg
     5521 tctgaaggtg aagagtccag agctttctcc tttgacgttg gtggtagagg ttacgttttg
     5581 agagttaatt cctgtgctga cggtttctac aaggacagat acgtttacag acacttcgct
     5641 tccgctgctt tgccaattcc agaggttttg gacattggtg agttctccga gtccttgact
     5701 tactgtatct ccagaagagc tcagggagtt acattgcaag acttgccaga aactgagttg
     5761 ccagctgttt tgcaaccagt tgctgaagct atggacgcta ttgctgctgc tgacttgtct
     5821 caaacttctg gttttggtcc attcggtcca cagggtattg gtcagtacac tacttggaga
     5881 gacttcatct gtgctatcgc tgacccacat gtttaccact ggcaaactgt tatggacgat
     5941 actgtttccg cttccgttgc tcaggctttg gacgaattga tgttgtgggc tgaagattgt
     6001 cctgaggtta gacatttggt tcacgctgac ttcggttcca acaacgtttt gactgacaac
     6061 ggtagaatca ctgctgttat cgactggtcc gaagctatgt tcggtgactc tcaatacgag
     6121 gttgctaaca tcttcttttg gagaccatgg ttggcttgta tggaacagca gactagatac
     6181 ttcgagagaa gacacccaga attggctggt tccccaagat tgagagctta catgttgaga
     6241 atcggtttgg accagttgta ccagtccttg gttgatggta acttcgatga tgctgcttgg
     6301 gctcaaggta gatgtgacgc tatcgttaga tccggtgctg gtactgttgg tagaactcag
     6361 attgctagaa gatccgctgc tgtttggact gacggttgtg ttgaagtttt ggctgactcc
     6421 ggtaacagaa gaccatccac tagaccaaga gctaaagagg ttggtagagt ttaagtatta
     6481 agaacgttat ttatatttca aatttttctt ttttttctct acagacgctg tacgcatgta
     6541 acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaagatc cagctgcatt
     6601 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6661 cggaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     6721 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     6781 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat
     6841 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     6901 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     6961 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     7021 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cga
//
