LOCUS       sequence-350972-        6462 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mWasabi for auxotrophic selection in the absence of 
            leucine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6462)
  AUTHORS   Yu JSL, Correia-Melo C, Zorrilla F, Herrera-Dominguez L, Wu MY, 
            Hartl J, Campbell K, Blasche S, Kreidl M, Egger AS, Messner CB, 
            Demichev V, Freiwald A, Mulleder M, Howell M, Berman J, Patil KR, 
            Alam MT, Ralser M
  TITLE     Microbial communities form rich extracellular metabolomes that 
            foster metabolic interactions and promote drug tolerance.
  JOURNAL   Nat Microbiol. 2022 Apr;7(4):542-555. doi: 
            10.1038/s41564-022-01072-5. Epub 2022 Mar 21.
  PUBMED    35314781
REFERENCE   2  (bases 1 to 6462)
  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"; doi: 
            "10.1038/s41564-022-01072-5"; journalName: "Nat Microbiol"; date: 
            "2022-04"; volume: "7"; issue: "4"; pages: "542-555"
COMMENT     Sequence Label: pL-mWasabi
FEATURES             Location/Qualifiers
     source          1..6462
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     139..158
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        177..843
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     713..736
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     terminator      1610..1797
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1665..1689)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     misc_feature    1833..2336
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        2786..3193
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             3194..4288
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(4354..4373)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4355..4373)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4460..4477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4631..5219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4711..4730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5390..6181,6182..6250))
                     /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     6013..6032
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6251..6355)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
       61 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
      121 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgtcat
      181 tatcaatact gccatttcaa agaatacgta aataattaat agtagtgatt ttcctaactt
      241 tatttagtca aaaaattagc cttttaattc tgctgtaacc cgtacatgcc caaaataggg
      301 ggcgggttac acagaatata taacatcgta ggtgtctggg tgaacagttt attcctggca
      361 tccactaaat ataatggagc ccgcttttta agctggcatc cagaaaaaaa aagaatccca
      421 gcaccaaaat attgttttct tcaccaacca tcagttcata ggtccattct cttagcgcaa
      481 ctacagagaa caggggcaca aacaggcaaa aaacgggcac aacctcaatg gagtgatgca
      541 acctgcctgg agtaaatgat gacacaaggc aattgaccca cgcatgtatc tatctcattt
      601 tcttacacct tctattacct tctgctctct ctgatttgga aaaagctgaa aaaaaaggtt
      661 gaaaccagtt ccctgaaatt attcccctac ttgactaata agtatataaa gacggtaggt
      721 attgattgta attctgtaaa tctatttctt aaacttctta aattctactt ttatagttag
      781 tctttttttt agttttaaaa caccaagaac ttagtttcga ataaacacac ataaacaaac
      841 aaagtcgacg gatcggtgac ggtgctggtt taattaacat ggtaagtaaa ggtgaagaga
      901 caactatggg ggtaatcaaa ccagacatga agatcaaact gaaaatggaa ggtaacgtta
      961 atggccacgc gtttgttatt gaaggtgaag gagaggggaa accttacgac ggaaccaaca
     1021 ctatcaattt ggaggttaag gaaggtgcac cattaccatt cagctatgac atcttaacaa
     1081 ctgcattttc atacggcaac agagctttta ccaagtaccc tgacgatata ccaaattact
     1141 tcaaacaatc ctttcctgaa ggttattcat gggagagaac aatgacgttt gaagataagg
     1201 gcattgtcaa ggtcaaatct gacatttcta tggaagagga ttcattcatc tacgagattc
     1261 acctcaaggg cgaaaacttc ccaccaaatg gtccagtgat gcaaaaggaa actactggat
     1321 gggatgcttc tacagaaagg atgtacgtga gagatggagt cttaaaaggt gatgttaaaa
     1381 tgaagttgct acttgaaggt ggtggccatc atagagttga tttcaaaaca atctacagag
     1441 ccaaaaaggc tgtaaaactt cctgattatc attttgtgga tcatcgtata gaaattctaa
     1501 accacgacaa agattacaat aaggtcactg tttatgaaat agcagttgcc agaaattcta
     1561 cagatggaat ggatgaattg tacaaataag gcgcgccact tctaaataag cgaatttctt
     1621 atgatttatg atttttatta ttaaataagt tataaaaaaa ataagtgtat acaaatttta
     1681 aagtgactct taggttttaa aacgaaaatt cttattcttg agtaactctt tcctgtaggt
     1741 caggttgctt tctcaggtat agtatgaggt cgctcttatt gaccacacct ctaccggcag
     1801 atccgctagg gataacaggg taatatagat ctgatcgctt gcctgtaact tacacgcgcc
     1861 tcgtatcttt taatgatgga ataatttggg aatttactct gtatttattt atttttatgt
     1921 tttgtatttg gattttagaa agtaaataaa gaaggtagaa gagttacgga atgaagaaaa
     1981 aaaaataaac aaaggtttaa aaaatttcaa caaaaagcgt actttacata tatatttatt
     2041 agacaagaaa agcagattaa atagatatac attcgattaa cgataagtaa aatgtaaaat
     2101 cacaggattt tcgtgtgtgg tcttctacac agacaagatg aaacaattcg gcattaatac
     2161 ctgagagcag gaagagcaag ataaaaggta gtatttgttg gcgatccccc tagagtcttt
     2221 tacatcttcg gaaaacaaaa actatttttt ctttaatttc tttttttact ttctattttt
     2281 aatttatata tttatattaa aaaatttaaa ttataattat ttttatagca cgtgatgaaa
     2341 agaaccctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     2401 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     2461 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     2521 gtcagcgggt gttggcggcg cgccatcgag gagaacttct agtatatcta catacctaat
     2581 attattgcct tattaaaaat ggaatcccaa caattacatc aaaatccaca ttctcttcaa
     2641 aatcaattgt cctgtacttc cttgttcatg tgtgttcaaa aacgttatat ttataggata
     2701 attatactct atttctcaac aagtaattgg ttgtttggcc gagcggtcta aggcgcctga
     2761 ttcaagaaat atcttgaccg cagttaactg tgggaatact caggtatcgt aagatgcaag
     2821 agttcgaatc tcttagcaac cattattttt ttcctcaaca taacgagaac acacaggggc
     2881 gctatcgcac agaatcaaat tcgatgactg gaaatttttt gttaatttca gaggtcgcct
     2941 gacgcatata cctttttcaa ctgaaaaatt gggagaaaaa ggaaaggtga gagcgccgga
     3001 accggctttt catatagaat agagaagcgt tcatgactaa atgcttgcat cacaatactt
     3061 gaagttgaca atattattta aggacctatt gttttttcca ataggtggtt agcaatcgtc
     3121 ttactttcta acttttctta cctcttacat ttcagcaata tatatatata tatttcaagg
     3181 atataccatt ctaatgtctg cccctaagaa gatcgtcgtt ttgccaggtg accacgttgg
     3241 tcaagaaatc acagccgaag ccattaaggt tcttaaagct atttctgatg ttcgttccaa
     3301 tgtcaagttc gatttcgaaa atcatttaat tggtggtgct gctatcgatg ctacaggtgt
     3361 tccacttcca gatgaggcgc tggaagcctc caagaaggct gatgccgttt tgttaggtgc
     3421 tgtgggtggt cctaaatggg gtaccggtag tgttagacct gaacaaggtt tactaaaaat
     3481 ccgtaaagaa cttcaattgt acgccaactt aagaccatgt aactttgcat ccgactctct
     3541 tttagactta tctccaatca agccacaatt tgctaaaggt actgacttcg ttgttgtcag
     3601 agaattagtg ggaggtattt actttggtaa gagaaaggaa gacgatggtg atggtgtcgc
     3661 ttgggatagt gaacaataca ccgttccaga agtgcaaaga atcacaagaa tggccgcttt
     3721 catggcccta caacatgagc caccattgcc tatttggtcc ttggataaag ctaatgtttt
     3781 ggcctcttca agattatgga gaaaaactgt ggaggaaacc atcaagaacg aattccctac
     3841 attgaaggtt caacatcaat tgattgattc tgccgccatg atcctagtta agaacccaac
     3901 ccacctaaat ggtattataa tcaccagcaa catgtttggt gatatcatct ccgatgaagc
     3961 ctccgttatc ccaggttcct tgggtttgtt gccatctgcg tccttggcct ctttgccaga
     4021 caagaacacc gcatttggtt tgtacgaacc atgccacggt tctgctccag atttgccaaa
     4081 gaataaggtc aaccctatcg ccactatctt gtctgctgca atgatgttga aattgtcatt
     4141 gaacttgcct gaagaaggta aggccattga agatgcagtt aaaaaggttt tggatgcagg
     4201 tatcagaact ggtgatttag gtggttccaa cagtaccacc gaagtcggtg atgctgtcgc
     4261 cgaagaagtt aagaaaatcc ttgcttaaga gctcgaattc atcgatgata tcagatccac
     4321 tagtggccta tgcggccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga
     4381 taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4441 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4501 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4561 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4621 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4681 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4741 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4801 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4861 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4921 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4981 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5041 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5101 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5161 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5221 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5281 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5341 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5401 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5461 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5521 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5581 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5641 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5701 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5761 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5821 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5881 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5941 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6001 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6061 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6121 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6181 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6241 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6301 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6361 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     6421 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cg
//
