LOCUS       sequence-359389-        8968 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GAL1p:T7RNAP(F11L;T613A):CYC1t.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8968)
  AUTHORS   Jensen ED, Laloux M, Lehka BJ, Pedersen LE, Jakociunas T, Jensen MK,
            Keasling JD
  TITLE     A synthetic RNA-mediated evolution system in yeast.
  JOURNAL   Nucleic Acids Res. 2021 Sep 7;49(15):e88. doi: 10.1093/nar/gkab472.
  PUBMED    34107026
REFERENCE   2  (bases 1 to 8968)
  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: "Nucleic 
            Acids Res."; date: "2021-09-7"; volume: "49(15):e88. doi"; pages: " 
            10.1093/nar/gkab472"
COMMENT     Sequence Label: pMLB10
FEATURES             Location/Qualifiers
     source          1..8968
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        191..472
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             473..1147
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(1250..1705)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1387..1408)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1599..1618
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1784..1949)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1870..1894
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        2189..2630
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    2189..2306
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     2601..2624
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             2660..2680
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      5375..5564
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5636..5653)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5807..6395)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5887..5906)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6566..7357,7358..7426))
                     /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     7189..7208
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7427..7531)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7558..8900
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(8521..8568)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     8941..8959
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatagatc aacgacatta ctatatatat aatataggaa gcatttaata gaacagcatc
      241 gtaatatatg tgtactttgc agttatgacg ccagatggca gtagtggaag atattcttta
      301 ttgaaaaata gcttgtcacc ttacgtacaa tcttgatccg gagcttttct ttttttgccg
      361 attaagaatt aattcggtcg aaaaaagaaa aggagagggc caagagggag ggcattggtg
      421 actattgagc acgtgagtat acgtgattaa gcacacaaag gcagcttgga gtatgtctgt
      481 tattaatttc acaggtagtt ctggtccatt ggtgaaagtt tgcggcttgc agagcacaga
      541 ggccgcagaa tgtgctctag attccgatgc tgacttgctg ggtattatat gtgtgcccaa
      601 tagaaagaga acaattgacc cggttattgc aaggaaaatt tcaagtcttg taaaagcata
      661 taaaaatagt tcaggcactc cgaaatactt ggttggcgtg tttcgtaatc aacctaagga
      721 ggatgttttg gctctggtca atgattacgg cattgatatc gtccaactgc atggagatga
      781 gtcgtggcaa gaataccaag agttcctcgg tttgccagtt attaaaagac tcgtatttcc
      841 aaaagactgc aacatactac tcagtgcagc ttcacagaaa cctcattcgt ttattccctt
      901 gtttgattca gaagcaggtg ggacaggtga acttttggat tggaactcga tttctgactg
      961 ggttggaagg caagagagcc ccgaaagctt acattttatg ttagctggtg gactgacgcc
     1021 agaaaatgtt ggtgatgcgc ttagattaaa tggcgttatt ggtgttgatg taagcggagg
     1081 tgtggagaca aatggtgtaa aagactctaa caaaatagca aatttcgtca aaaatgctaa
     1141 gaaataggtt attactgagt agtatttatt taagtattgt ttgtgcactt gccgatctat
     1201 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     1261 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     1321 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     1381 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     1441 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     1501 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     1561 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     1621 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     1681 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     1741 ggcgatcggt gcgggcctct tcgctattac gccagctgaa ttggagcgac ctcatgctat
     1801 acctgagaaa gcaacctgac ctacaggaaa gagttactca agaataagaa ttttcgtttt
     1861 aaaacctaag agtcacttta aaatttgtat acacttattt tttttataac ttatttaata
     1921 ataaaaatca taaatcataa gaaattcgct tatttagaag tgtcaacaac gtatctacca
     1981 acgatttgac ccttttccat cttttcgtaa atttctggca aggtagacaa gccgacaacc
     2041 ttgattggag acttgaccaa acctctggcg aagaattgtt aattaagagc tcgaatgcgt
     2101 gcgatgagcc ccattatctt agcctaaaaa aaccttctct ttggaacttt cagtaatacg
     2161 cttaactgct cattgctata ttgaagtacg gattagaagc cgccgagcgg gcgacagccc
     2221 tccgacggaa gactctcctc cgtgcgtcct ggtcttcacc ggtcgcgttc ctgaaacgca
     2281 gatgtgcctc gcgccgcact gctccgaaca ataaagattc tacaatacta gcttttatgg
     2341 ttatgaagag gaaaaattgg cagtaacctg gccccacaaa ccttcaaatc aacgaatcaa
     2401 attaacaacc ataggataat aatgcgatta gttttttagc cttatttctg gggtaattaa
     2461 tcagcgaagc gatgattttt gatctattaa cagatatata aatgcaaaag ctgcataacc
     2521 actttaacta atactttcaa cattttcggt ttgtattact tcttattcaa atgtcataaa
     2581 agtatcaaca aaaaattgtt aatatacctc tatactttaa cgtcaaggag aaaaaactat
     2641 aatctgtcat aaaacaatgc ccaagaagaa gaggaaggtg aacacgatta acatcgctaa
     2701 gaacgacttg tctgacatcg aactggctgc tatcccgttc aacactctgg ctgaccatta
     2761 cggtgagcgt ttagctcgcg aacagttggc ccttgagcat gagtcttacg agatgggtga
     2821 agcacgcttc cgcaagatgt ttgagcgtca acttaaagct ggtgaggttg cggataacgc
     2881 tgccgccaag cctctcatca ctaccctact ccctaagatg attgcacgca tcaacgactg
     2941 gtttgaggaa gtgaaagcta agcgcggcaa gcgcccgaca gccttccagt tcctgcaaga
     3001 aatcaagccg gaagccgtag cgtacatcac cattaagacc actctggctt gcctaaccag
     3061 tgctgacaat acaaccgttc aggctgtagc aagcgcaatc ggtcgggcca ttgaggacga
     3121 ggctcgcttc ggtcgtatcc gtgaccttga agctaagcac ttcaagaaaa acgttgagga
     3181 acaactcaac aagcgcgtag ggcacgtcta caagaaagca tttatgcaag ttgtcgaggc
     3241 tgacatgctc tctaagggtc tactcggtgg cgaggcgtgg tcttcgtggc ataaggaaga
     3301 ctctattcat gtaggagtac gctgcatcga gatgctcatt gagtcaaccg gaatggttag
     3361 cttacaccgc caaaatgctg gcgtagtagg tcaagactct gagactatcg aactcgcacc
     3421 tgaatacgct gaggctatcg caacccgtgc aggtgcgctg gctggcatct ctccgatgtt
     3481 ccaaccttgc gtagttcctc ctaagccgtg gactggcatt actggtggtg gctattgggc
     3541 taacggtcgt cgtcctctgg cgctggtgcg tactcacagt aagaaagcac tgatgcgcta
     3601 cgaagacgtt tacatgcctg aggtgtacaa agcgattaac attgcgcaaa acaccgcatg
     3661 gaaaatcaac aagaaagtcc tagcggtcgc caacgtaatc accaagtgga agcattgtcc
     3721 ggtcgaggac atccctgcga ttgagcgtga agaactcccg atgaaaccgg aagacatcga
     3781 catgaatcct gaggctctca ccgcgtggaa acgtgctgcc gctgctgtgt accgcaagga
     3841 caaggctcgc aagtctcgcc gtatcagcct tgagttcatg cttgagcaag ccaataagtt
     3901 tgctaaccat aaggccatct ggttccctta caacatggac tggcgcggtc gtgtttacgc
     3961 tgtgtcaatg ttcaacccgc aaggtaacga tatgaccaaa ggactgctta cgctggcgaa
     4021 aggtaaacca atcggtaagg aaggttacta ctggctgaaa atccacggtg caaactgtgc
     4081 gggtgtcgat aaggttccgt tccctgagcg catcaagttc attgaggaaa accacgagaa
     4141 catcatggct tgcgctaagt ctccactgga gaacacttgg tgggctgagc aagattctcc
     4201 gttctgcttc cttgcgttct gctttgagta cgctggggta cagcaccacg gcctgagcta
     4261 taactgctcc cttccgctgg cgtttgacgg gtcttgctct ggcatccagc acttctccgc
     4321 gatgctccga gatgaggtag gtggtcgcgc ggttaacttg cttcctagtg aaaccgttca
     4381 ggacatctac gggattgttg ctaagaaagt caacgagatt ctacaagcag acgcaatcaa
     4441 tgggaccgat aacgaagtag ttaccgtgac cgatgagaac actggtgaaa tctctgagaa
     4501 agtcaagctg ggcgctaagg cactggctgg tcaatggctg gcttacggtg ttactcgcag
     4561 tgtgactaag cgttcagtca tgacgctggc ttacgggtcc aaagagttcg gcttccgtca
     4621 acaagtgctg gaagatacca ttcagccagc tattgattcc ggcaagggtc tgatgttcac
     4681 tcagccgaat caggctgctg gatacatggc taagctgatt tgggaatctg tgagcgtgac
     4741 ggtggtagct gcggttgaag caatgaactg gcttaagtct gctgctaagc tgctggctgc
     4801 tgaggtcaaa gataagaaga ctggagagat tcttcgcaag cgttgcgctg tgcattgggt
     4861 aactcctgat ggtttccctg tgtggcagga atacaagaag cctattcaga cgcgcttgaa
     4921 cctgatgttc ctcggtcagt tccgcttaca gcctaccatt aacaccaaca aagatagcga
     4981 gattgatgca cacaaacagg agtctggtat cgctcctaac tttgtacaca gccaagacgg
     5041 tagccacctt cgtaagactg tagtgtgggc acacgagaag tacggaatcg aatcttttgc
     5101 actgattcac gactccttcg gtaccattcc ggctgacgct gcgaacctgt tcaaagcagt
     5161 gcgcgaaact atggttgaca catatgagtc ttgtgatgta ctggctgatt tctacgacca
     5221 gttcgctgac cagttgcacg agtctcaatt ggacaaaatg ccagcacttc cggctaaagg
     5281 taacttgaac ctccgtgaca tcttagagtc ggacttcgcg ttcgcgtaaa tcgcgtgcat
     5341 tcctcgagta agcttggtac cgcggctagc taagatccgc tctaaccgaa aaggaaggag
     5401 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     5461 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
     5521 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaagatccag ctgcattaat
     5581 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5641 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5701 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5761 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5821 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5881 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5941 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6001 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6061 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6121 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6181 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6241 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6301 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6361 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6421 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6481 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6541 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6601 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6661 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6721 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6781 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6841 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6901 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6961 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7021 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7081 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7141 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7201 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7261 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7321 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7381 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7441 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7501 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac
     7561 gaagcatctg tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa
     7621 acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac
     7681 caacgaagaa tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt
     7741 ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat
     7801 tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct
     7861 atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag
     7921 tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt
     7981 ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg
     8041 aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg
     8101 gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa
     8161 attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt
     8221 tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag
     8281 taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc
     8341 gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac
     8401 ttttgagcaa tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg
     8461 tgcgtttttg gttttttgaa agtgcgtctt cagagcgctt ttggttttca aaagcgctct
     8521 gaagttccta tactttctag agaataggaa cttcggaata ggaacttcaa agcgtttccg
     8581 aaaacgagcg cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc
     8641 gcacctatat ctgcgtgttg cctgtatata tatatacatg agaagaacgg catagtgcgt
     8701 gtttatgctt aaatgcgtac ttatatgcgt ctatttatgt aggatgaaag gtagtctagt
     8761 acctcctgtg atattatccc attccatgcg gggtatcgta tgcttccttc agcactaccc
     8821 tttagctgtt ctatatgctg ccactcctca attggattag tctcatcctt caatgctatc
     8881 atttcctttg atattggatc atactaagaa accattatta tcatgacatt aacctataaa
     8941 aataggcgta tcacgaggcc ctttcgtc
//
