LOCUS       sequence-484589-        6647 bp    DNA     circular SYN 25-AUG-2025
DEFINITION  Backbone for PYR1 sensor library assembly from oligopools; 
            domesticated for golden gate cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6647)
  AUTHORS   Tian H, Beltran J, George W, Lenert-Mondou C, Seder N, Davis ZI, 
            Swift SD, Girke T, Whitehead TA, Wheeldon I, Cutler SR
  TITLE     Unusually Broad-spectrum small-molecule sensing using a single 
            protein scaffold.
  JOURNAL   bioRxiv [Preprint]. 2025 May 15:2025.05.15.654352. doi: 
            10.1101/2025.05.15.654352.
  PUBMED    40462935
REFERENCE   2  (bases 1 to 6647)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 May 15:2025.05.15.654352. doi: 
            10.1101/2025.05.15.654352."
COMMENT     Sequence Label: pBD_PYR1_156
FEATURES             Location/Qualifiers
     source          1..6647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..406
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             434..874
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     primer_bind     complement(1072..1091)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1073..1091)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      1113..1300
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1168..1192)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(1353..2027)
                     /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"
     promoter        complement(2028..2129)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(2143..2159)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2143..2159)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2156..2178)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2167..2183
                     /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)."
     promoter        complement(join(2191..2197,2198..2215,2216..2221))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2236..2257
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        complement(2329..2433)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2460..2915
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2547..2566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2757..2778
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3010..3027)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3181..3769)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3261..3280)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4226..4328
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4329..4988
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4395..4414)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     5269..5287
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5325..5347)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5447..5466
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      5483..6647
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 gcttgcatgc aacttctttt cttttttttt cttttctctc tcccccgttg ttgtctcacc
       61 atatccgcaa tgacaaaaaa atgatggaag acactaaagg aaaaaattaa cgacaaagac
      121 agcaccaaca gatgtcgttg ttccagagct gatgaggggt atctcgaagc acacgaaact
      181 ttttccttcc ttcattcacg cacactactc tctaatgagc aacggtatac ggccttcctt
      241 ccagttactt gaatttgaaa taaaaaaaag tttgctgtct tgctatcaag tataaataga
      301 cctgcaatta ttaatctttt gtttcctcgt cattgttctc gttccctttc ttccttgttt
      361 ctttttctgc acaatatttc aagctatacc aagcatacaa tcaactccaa gcttgaagca
      421 agcctcctga aagatgaagc tactgtcttc tatcgaacaa gcatgcgata tttgccgact
      481 taaaaagctc aagtgctcca aagaaaaacc gaagtgcgcc aagtgtctga agaacaactg
      541 ggagtgtcgc tactctccca aaaccaaaag gtccccgctg actagggcac atctgacaga
      601 agtggaatca aggctagaaa gactggaaca gctatttcta ctgatttttc ctcgagaaga
      661 ccttgacatg attttgaaaa tggattcttt acaggatata aaagcattgt taacaggatt
      721 atttgtacaa gataatgtga ataaagatgc cgtcacagat agattggctt cagtggagac
      781 tgatatgcct ctaacattga gacagcatag aataagtgcg acatcatcat cggaagagag
      841 tagtaacaaa ggtcaaagac agttgactgt atcgccggaa ttcatgcctt cggagttaac
      901 accagaagaa cgatcggaac taaaaaactc aatcgccgag ttccacacat accaactcga
      961 tccaggaagc tgttcatcac tccacgcgca acgaatccac gcgcctccgg aactcgtctg
     1021 gtcaatcgta cgacgattcg gagacctcga ggtctcgggt cgactctaga gccctatagt
     1081 gagtcgtatt actgcagcca agctaattcc gggcgaattt cttatgattt atgattttta
     1141 ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt
     1201 taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg
     1261 tatagcatga ggtcgctctt attgaccaca cctctaccgg catgccggca agtgcacaaa
     1321 caatacttaa ataaatacta ctcagtaata acctatttct tagcattttt gacgaaattt
     1381 gctattttgt tagagtcttt tacaccattt gtctccacac ctccgcttac atcaacacca
     1441 ataacgccat ttaatctaag cgcatcacca acattttctg gcgtcagtcc accagctaac
     1501 ataaaatgta agctttcggg gctctcttgc cttccaaccc agtcagaaat cgagttccaa
     1561 tccaaaagtt cacctgtccc acctgcttct gaatcaaaca agggaataaa cgaatgaggt
     1621 ttctgtgaag ctgcactgag tagtatgttg cagtcttttg gaaatacgag tcttttaata
     1681 actggcaaac cgaggaactc ttggtattct tgccacgact catctccatg cagttggacg
     1741 atatcaatgc cgtaatcatt gaccagagcc aaaacatcct ccttaggttg attacgaaac
     1801 acgccaacca agtatttcgg agtgcctgaa ctatttttat atgcttttac aagacttgaa
     1861 attttccttg caataaccgg gtcaattgtt ctctttctat tgggcacaca tataataccc
     1921 agcaagtcag catcggaatc tagagcacat tctgcggcct ctgtgctctg caagccgcaa
     1981 actttcacca atggaccaga actacctgtg aaattaataa cagacatact ccaagctgcc
     2041 tttgtgtgct taatcacgta tactcacgtg ctcaatagtc accaatgccc tccctcttgg
     2101 ccctctcctt ttcttttttc gaccgaatta attcgtaatc atgtcatagc tgtttcctgt
     2161 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2221 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     2281 tttccagtcg ggaaacctgt cgtgccagct ggttctttcc gcctcaggac tcttcctttt
     2341 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     2401 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     2461 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2521 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2581 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2641 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2701 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2761 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2821 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2881 cgcgaatttt aacaaaatat taacgcttac aatttacgcg cctaaattat gcggccgcat
     2941 ataactgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3001 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3061 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3121 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3181 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3241 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3301 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3361 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3421 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3481 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3541 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3601 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3661 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3721 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3781 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3841 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3901 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3961 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4021 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4081 ggacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg cctgtgacgg
     4141 aagatcactt cgcagaataa ataaatcctg gtgtccctgt tgataccggg aagccctggg
     4201 ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag aggttccaac tttcaccata
     4261 atgaaataag atcactaccg ggcgtatttt ttgagttgtc gagattttca ggagctaagg
     4321 aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc
     4381 gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc
     4441 agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg
     4501 cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattacgt atggcaatga
     4561 aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc
     4621 aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac
     4681 acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt
     4741 ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt
     4801 taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata
     4861 cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg
     4921 gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg
     4981 gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggttgc tacgcctgaa
     5041 taagtgataa taagcggatg aatggcagaa attcgaaagc aaattcgacc cggtcgtcgg
     5101 ttcagggcag ggtcgttaaa tagccgctta tgtctattgc tggtttaccg gtttattgac
     5161 taccggaagc agtgtgaccg tgtgcttctc aaatgcctga ggccagtttg ctcaggctct
     5221 ccccgtggag gctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     5281 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     5341 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     5401 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     5461 attgtactga gagtgcacca taacgcattt aagcataaac acgcactatg ccgttcttct
     5521 catgtatata tatatacagg caacacgcag atataggtgc gacgtgaaca gtgagctgta
     5581 tgtgcgcagc tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct ttgaagttcc
     5641 tattccgaag ttcctattct ctagctagaa agtataggaa cttcagagcg cttttgaaaa
     5701 ccaaaagcgc tctgaagacg cactttcaaa aaaccaaaaa cgcaccggac tgtaacgagc
     5761 tactaaaata ttgcgaatac cgcttccaca aacattgctc aaaagtatct ctttgctata
     5821 tatctctgtg ctatatccct atataaccta cccatccacc tttcgctcct tgaacttgca
     5881 tctaaactcg acctctacat tttttatgtt tatctctagt attactcttt agacaaaaaa
     5941 attgtagtaa gaactattca tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc
     6001 tatacgtagt atatagagac aaaatagaag aaaccgttca taattttctg accaatgaag
     6061 aatcatcaac gctatcactt tctgttcaca aagtatgcgc aatccacatc ggtatagaat
     6121 ataatcgggg atgcctttat cttgaaaaaa tgcacccgca gcttcgctag taatcagtaa
     6181 acgcgggaag tggagtcagg ctttttttat ggaagagaaa atagacacca aagtagcctt
     6241 cttctaacct taacggacct acagtgcaaa aagttatcaa gagactgcat tatagagcgc
     6301 acaaaggaga aaaaaagtaa tctaagatgc tttgttagaa aaatagcgct ctcgggatgc
     6361 atttttgtag aacaaaaaag aagtatagat tctttgttgg taaaatagcg ctctcgcgtt
     6421 gcatttctgt tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta gcgctctcgc
     6481 gttgcatttt tgttttacaa aaatgaagca cagattcttc gttggtaaaa tagcgctttc
     6541 gcgttgcatt tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa aattagcgct
     6601 ctcgcgttgc atttttgttc tacaaaatga agcacagatg cttcgtt
//
