LOCUS       sequence-457120-        7237 bp DNA     circular SYN 28-MAR-2025
DEFINITION  Doxycycline-regulatable EGFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7237)
  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 7237)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 28, 2025 from SnapGene Server 7.0.3
            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"
FEATURES             Location/Qualifiers
     source          1..7237
                     /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             201..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        2144..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"
     CDS             4174..4890
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      4968..5214
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             5611..6633
                     /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(6843..6860)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6843..6859)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6852..6874)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(7068..7087)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7187..7209
                     /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 gtggatgata atgctatcga tagcaatata taaacagaag
     4021 gaagctgccc tgtcttaaac cttttttttt atcatcatta ttagcttact ttcataattg
     4081 cgactggttc caattgacaa gcttttgatt ttaacgactt ttaacgacaa cttgagaaga
     4141 tcaaaaaaca actaattatt cgaaacgact agtatggttt ctaagggtga agagttgttc
     4201 actggtgttg ttccaatctt ggttgagttg gacggtgacg ttaacggaca caagttctct
     4261 gtttctggtg aaggtgaggg tgacgctact tacggaaagt tgactttgaa gttcatctgt
     4321 actactggta agttgccagt tccatggcca actttggtta ctactttgac ttacggtgtt
     4381 cagtgtttct ccagataccc agaccacatg aagcagcacg atttcttcaa gtctgctatg
     4441 ccagagggtt acgttcaaga gagaactatc ttcttcaagg acgacggtaa ctacaagact
     4501 agagctgagg ttaagttcga gggtgacaca ttggttaaca gaatcgagtt gaagggtatc
     4561 gacttcaaag aggacggaaa catcttgggt cacaagttgg agtacaacta caactcccac
     4621 aacgtttaca tcatggctga caagcagaag aacggtatca aggttaactt caagatcaga
     4681 cacaacatcg aggacggttc cgttcaattg gctgaccact accaacagaa cactccaatt
     4741 ggtgacggtc cagttttgtt gccagacaac cactacttgt ccactcaatc cgctttgtcc
     4801 aaggacccaa acgagaagag agatcacatg gttttgttgg agttcgttac tgctgctggt
     4861 atcactttgg gtatggacga gttgtacaag taactcgagc cttagacatg actgttcctc
     4921 agttcaagtt gggcacttac gagaagaccg gtcttgctag attctaatca agaggatgtc
     4981 agaatgccat ttgcctgaga gatgcaggct tcatttttga tactttttta tttgtaacct
     5041 atatagtata ggattttttt tgtcattttg tttcttctcg tacgagcttg ctcctgatca
     5101 gcctatctcg cagctgatga atatcttgtg gtaggggttt gggaaaatca ttcgagtttg
     5161 atgtttttct tggtatttcc cactcctctt cagagtacag aagattaagt gagacgttcg
     5221 tttgtgcaag cttagatccc cgggtaccga gctcgaattc gccgtaccac ttcaaaacac
     5281 ccaagcacag catactaaat ttcccctctt tcttcctcta gggtgtcgtt aattacccgt
     5341 actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg
     5401 caataaaaat ttttatcacg tttctttttc ttgaaaattt ttttttttga tttttttctc
     5461 tttcgatgac ctcccattga tatttaagtt aataaacggt cttcaatttc tcaagtttca
     5521 gtttcatttt tcttgttcta ttacaacttt ttttacttct tgctcattag aaagaaagca
     5581 tagcaatcta atctaagttt taattacaaa atgaagaagc cagagttgac tgctacttcc
     5641 gttgagaagt tcttgatcga gaagttcgac tccgtttccg acttgatgca attgtctgaa
     5701 ggtgaagagt ccagagcttt ctcctttgac gttggtggta gaggttacgt tttgagagtt
     5761 aattcctgtg ctgacggttt ctacaaggac agatacgttt acagacactt cgcttccgct
     5821 gctttgccaa ttccagaggt tttggacatt ggtgagttct ccgagtcctt gacttactgt
     5881 atctccagaa gagctcaggg agttacattg caagacttgc cagaaactga gttgccagct
     5941 gttttgcaac cagttgctga agctatggac gctattgctg ctgctgactt gtctcaaact
     6001 tctggttttg gtccattcgg tccacagggt attggtcagt acactacttg gagagacttc
     6061 atctgtgcta tcgctgaccc acatgtttac cactggcaaa ctgttatgga cgatactgtt
     6121 tccgcttccg ttgctcaggc tttggacgaa ttgatgttgt gggctgaaga ttgtcctgag
     6181 gttagacatt tggttcacgc tgacttcggt tccaacaacg ttttgactga caacggtaga
     6241 atcactgctg ttatcgactg gtccgaagct atgttcggtg actctcaata cgaggttgct
     6301 aacatcttct tttggagacc atggttggct tgtatggaac agcagactag atacttcgag
     6361 agaagacacc cagaattggc tggttcccca agattgagag cttacatgtt gagaatcggt
     6421 ttggaccagt tgtaccagtc cttggttgat ggtaacttcg atgatgctgc ttgggctcaa
     6481 ggtagatgtg acgctatcgt tagatccggt gctggtactg ttggtagaac tcagattgct
     6541 agaagatccg ctgctgtttg gactgacggt tgtgttgaag ttttggctga ctccggtaac
     6601 agaagaccat ccactagacc aagagctaaa gaggttggta gagtttaagt attaagaacg
     6661 ttatttatat ttcaaatttt tctttttttt ctctacagac gctgtacgca tgtaacatta
     6721 tactgaaaac cttgcttgag aaggttttgg gacgctcgaa gatccagctg cattaatgaa
     6781 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcggaat
     6841 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     6901 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     6961 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     7021 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     7081 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     7141 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     7201 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga
//
