LOCUS       sequence-457104-        6124 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of mutant rTetTA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6124)
  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 6124)
  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: pMT1375
FEATURES             Location/Qualifiers
     source          1..6124
                     /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"
     promoter        2256..2732
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             join(2756..3373,3374..3379,3380..3415,3416..3418,3419..3454,
                     3455..3457,3458..3493,3494..3502)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSNVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(4643..5437)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4754..4773)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5364..5383
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(5730..5747)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5730..5746)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5739..5761)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5955..5974)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6074..6096
                     /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 cgccaagctt gcggccgcgg atcctttttt gtagaaatgt cttggtgtcc
     2281 tcgtccaatc aggtagccat ctctgaaata tctggctccg ttgcaactcc gaacgacctg
     2341 ctggcaacgt aaaattctcc ggggtaaaac ttaaatgtgg agtaatggaa ccagaaacgt
     2401 ctcttccctt ctctctcctt ccaccgcccg ttaccgtccc taggaaattt tactctgctg
     2461 gagagcttct tctacggccc ccttgcagca atgctcttcc cagcattacg ttgcgggtaa
     2521 aacggaggtc gtgtacccga cctagcagcc cagggatgga aaagtcccgg ccgtcgctgg
     2581 caataatagc gggcggacgc atgtcatgag attattggaa accaccagaa tcgaatataa
     2641 aaggcgaaca cctttcccaa ttttggtttc tcctgaccca aagactttaa atttaattta
     2701 tttgtcccta tttcaatcaa ttgaacaact atcaaaacac aactagtgcg tcgacatgtc
     2761 tagactggac aagagcaatg tcataaacgg cgctctggaa ttactcaatg gagtcggtat
     2821 cgaaggcctg acgacaagga aactcgctca aaagctggga gttgagcagc ctaccctgta
     2881 ctggcacgtg aagaacaagc gggccctgct cgatgccctg ccaatcgaga tgctggacag
     2941 gcatcatacc cacttctgcc ccctggaagg cgagtcatgg caagactttc tgcggaacaa
     3001 cgccaagtca ttccgctgtg ctctcctctc acatcgcgac ggggctaaag tgcatctcgg
     3061 cacccgccca acagagaaac agtacgaaac cctggaaaat cagctcgcgt tcctgtgtca
     3121 gcaaggcttc tccctggaga acgcactata cgctctgtcc gccgtgggcc actttacact
     3181 gggctgcgta ttggaggaac aggagcatca agtagcaaaa gaggaaagag agacacctac
     3241 caccgattct atgcccccac ttctgagaca agcaattgag ctgttcgacc ggcagggagc
     3301 cgaacctgcc ttccttttcg gcctggaact aatcatatgt ggcctggaga aacagctaaa
     3361 gtgcgaaagc ggcgggccgg ccgacgccct tgacgatttt gacttagaca tgctcccagc
     3421 cgatgccctt gacgactttg accttgatat gctgcctgct gacgctcttg acgattttga
     3481 ccttgacatg ctccccgggt aactaacctc gagtcaagga gttcattatt caagtctata
     3541 acccttcaaa gttgaaagtt cacgcagtaa acaccaacaa caaacgcaga tcaaaactaa
     3601 attgctctct ctcattccaa aacacattaa tcagctcttt gcaagactac acagacaatc
     3661 tctatggatg ctttattagc gggaacttgg agattcccgg caagtatcta ttacaagttc
     3721 ataaaaacat tatatctaac gaagaatgtt tggtcgaaat tggatctagt tcgtcatttg
     3781 agttatggga acatcattaa acaaacttct tatctaccat ctctgggtat ttttcaataa
     3841 catagtccaa gtccttatca cctctaccac ttagattgac caaaatggtt tgatcctttg
     3901 gtagagtcct ggccagcttg atagcataag cgatggcatg actggactct aaggctggaa
     3961 tgataccttc taatttggac agcatgaaga aagcttcaac agcttcattg tcaagatctt
     4021 ctagagacaa taagaagaaa aaaaaagaaa agcggtgggg gagggattat taaataagga
     4081 ttatgtaacc ccagggtacc gttctataca tatttaagga ttatttagga caatcgatga
     4141 aatcggcatc aaactggatg ggagtatagt gtccggataa tcggataaat catcttgcga
     4201 ggagccgctt ggttggttgg tgagaggagt gaaatatgtg tctcctcacc caagaatcgc
     4261 gatatcagca ccctgtgggg gacactattg gcctccctcc caaaccttcg atgtggtagt
     4321 gctttattat attgattaca ttgattacat agctaaaccc tgcctggttg caagttgagc
     4381 tccgaattcc aatattagta aaatgcctgc aagataacct cggtatggcg tccgaccccg
     4441 cttaattatt ttaactcctt tccaacgagg acttcgtaat ttttgattag ggagttgaga
     4501 aacggggggt cttgatacct cctcgatttc agatcccacc ccctctcagt cccaagtggg
     4561 acccccctcg gccgtgaaat gcgcgcactt tagttttttt cgcatgtaaa cgccggtgtc
     4621 cgtcatctag agaattcccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc
     4681 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc
     4741 caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac
     4801 ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca
     4861 agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgcgc gccttgagcc
     4921 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
     4981 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
     5041 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
     5101 ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata
     5161 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg
     5221 tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca
     5281 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
     5341 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg
     5401 ccggagaacc tgcgtgcaat ccatcttgtt caatcatatg gtttctatat tatctttgta
     5461 ctaaagagca attgataatg tgcgagaaaa actggtcctt atataccgtt tgcagcactc
     5521 cctcccgaac tttacgaaaa gtcgtgcgcc acctgatttt catcacgcca aaaacctaca
     5581 cgtatgacta ctccgggcca gtgttcacca cgagctatat agtgttaatt aattacctta
     5641 ttggttagct ctgcatgtaa gggtggtgtg agccgggaat tgggtctact ctagcgttca
     5701 gtaaggtgat ataaagctct gtagaattca ctggccgtcg ttttacaacg tcgtgactgg
     5761 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     5821 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     5881 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5941 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     6001 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     6061 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     6121 gcga
//
