LOCUS       sequence-457110-        5960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive mutant rPhlTA expression with strong promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5960)
  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 5960)
  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: pMT899
FEATURES             Location/Qualifiers
     source          1..5960
                     /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             3485..3505
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      3584..3830
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(4479..5273)
                     /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(4590..4609)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5200..5219
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(5566..5583)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5566..5582)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5575..5597)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5791..5810)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5910..5932
                     /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 tcgacatggc
     2761 tagaacttca cccagatcat ctattggttc tctgagatca ccacatactc ataaggctat
     2821 tttgacctcc accatcgaaa tcttgaaaga atgtggttac tccggtttgt ccattgaatc
     2881 tgttgctaga cgtgctggtg cttctaaacc tactatctac agatggtgga ctaacaaggc
     2941 tgctttgatt gctgaagttt acgaaaacga atccgaacag gttagaaagt ttccagattt
     3001 gggttccttt acggccgatt tggatttctt gttgagaaac ttgtggaagg tttggagaga
     3061 aactatttgt ggtgaagccc tcagatgcgt tattgcagaa gctcgattag atccagctac
     3121 tttgactcaa ttgaaggacc aattcatgga aagacgtaga gaaatgccaa aaaagttggt
     3181 taaaaacgcc atctctaacg gtgaattgcc aaaggataca aaccgtgagt tgttgttgga
     3241 tatgattttc ggtttttgct ggtacaggtt gttgactgaa caattgactg ttgaacaaga
     3301 catcgaagag ttcacgttct tgttaatcaa tggtgtttgt ccaggtactc aaagaggtcc
     3361 agcagacgct ttggatgatt ttgatttgga tatgttgcct gctgatgcct tggatgactt
     3421 cgacttagac atgttaccag ccgatgcatt agacgatttc gaccttgata tgttacctgg
     3481 tccaccaaaa aagaagagaa aggtctaacc tcgagcctta gacatgactg ttcctcagtt
     3541 caagttgggc acttacgaga agaccggtct tgctagattc taatcaagag gatgtcagaa
     3601 tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg taacctatat
     3661 agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc tgatcagcct
     3721 atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg agtttgatgt
     3781 ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga cgttcgtttg
     3841 tgcaagctta gatcttctag agacaataag aagaaaaaaa aagaaaagcg gtgggggagg
     3901 gattattaaa taaggattat gtaaccccag ggtaccgttc tatacatatt taaggattat
     3961 ttaggacaat cgatgaaatc ggcatcaaac tggatgggag tatagtgtcc ggataatcgg
     4021 ataaatcatc ttgcgaggag ccgcttggtt ggttggtgag aggagtgaaa tatgtgtctc
     4081 ctcacccaag aatcgcgata tcagcaccct gtgggggaca ctattggcct ccctcccaaa
     4141 ccttcgatgt ggtagtgctt tattatattg attacattga ttacatagct aaaccctgcc
     4201 tggttgcaag ttgagctccg aattccaata ttagtaaaat gcctgcaaga taacctcggt
     4261 atggcgtccg accccgctta attattttaa ctcctttcca acgaggactt cgtaattttt
     4321 gattagggag ttgagaaacg gggggtcttg atacctcctc gatttcagat cccaccccct
     4381 ctcagtccca agtgggaccc ccctcggccg tgaaatgcgc gcactttagt ttttttcgca
     4441 tgtaaacgcc ggtgtccgtc atctagagaa ttccccgctc agaagaactc gtcaagaagg
     4501 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
     4561 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
     4621 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
     4681 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc
     4741 atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
     4801 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
     4861 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
     4921 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc
     4981 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct
     5041 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca
     5101 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc
     5161 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc
     5221 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catatggttt
     5281 ctatattatc tttgtactaa agagcaattg ataatgtgcg agaaaaactg gtccttatat
     5341 accgtttgca gcactccctc ccgaacttta cgaaaagtcg tgcgccacct gattttcatc
     5401 acgccaaaaa cctacacgta tgactactcc gggccagtgt tcaccacgag ctatatagtg
     5461 ttaattaatt accttattgg ttagctctgc atgtaagggt ggtgtgagcc gggaattggg
     5521 tctactctag cgttcagtaa ggtgatataa agctctgtag aattcactgg ccgtcgtttt
     5581 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     5641 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     5701 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     5761 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5821 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5881 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5941 gtcatcaccg aaacgcgcga
//
