LOCUS       sequence-319292-        6373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Wild type VanR where the residue in position 165 was mutated from 
            Phenylalanine to Arginine. This mutation abolished the response to 
            vanillic acid. The gene is under the control of the TEF1 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6373)
  AUTHORS   D'Ambrosio, V., Pramanik, S., Goroncy, K., Jakociunas, T., 
            Schonauer, D., Davari, H. D., Schwaneberg, U., Keasling, J.D., 
            Jensen, M.K.
  TITLE     Directed evolution of VanR biosensor specificity in yeast
  JOURNAL   Biotechnology Notes, Volume 1, 2020, Pages 9-15
REFERENCE   2  (bases 1 to 6373)
  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: 
            "Biotechnology Notes, Volume 1, 2020, Pages 9-15"
COMMENT     Sequence Label: VanR - F165R
FEATURES             Location/Qualifiers
     source          1..6373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    60..563
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     604..622
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(660..682)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     782..801
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        825..1040
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             1041..1844
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1975..2430)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2112..2133)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2324..2343
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2556..2578
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2570..2587
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2571..2587
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2597..2616
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2597..2615
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(2644..2809)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2730..2754
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        2858..3264
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     complement(4231..4251)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4231..4249)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4270..4286)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4270..4286)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4283..4305)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4294..4310
                     /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(4318..4324,4325..4342,4343..4348))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4363..4384
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4501..4518)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4672..5260)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4752..4771)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5431..6222,6223..6291))
                     /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     6054..6073
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6292..6373,1..23))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgg gtccttttca
       61 tcacgtgcta taaaaataat tataatttaa attttttaat ataaatatat aaattaaaaa
      121 tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg ttttccgaag atgtaaaaga
      181 ctctaggggg atcgccaaca aatactacct tttatcttgc tcttcctgct ctcaggtatt
      241 aatgccgaat tgtttcatct tgtctgtgta gaagaccaca cacgaaaatc ctgtgatttt
      301 acattttact tatcgttaat cgaatgtata tctatttaat ctgcttttct tgtctaataa
      361 atatatatgt aaagtacgct ttttgttgaa attttttaaa cctttgttta tttttttttc
      421 ttcattccgt aactcttcta ccttctttat ttactttcta aaatccaaat acaaaacata
      481 aaaataaata aacacagagt aaattcccaa attattccat cattaaaaga tacgaggcgc
      541 gtgtaagtta caggcaagcg atccgtctaa gaaaccatta ttatcatgac attaacctat
      601 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
      661 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
      721 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
      781 gcggcatcag agcagattgt actgagagtg caccatacca ccttttcaat tcatcatttt
      841 ttttttattc ttttttttga tttcggtttc cttgaaattt ttttgattcg gtaatctccg
      901 aacagaagga agaacgaagg aaggagcaca gacttagatt ggtatatata cgcatatgta
      961 gtgttgaaga aacatgaaat tgcccagtat tcttaaccca actgcacaga acaaaaacct
     1021 gcaggaaacg aagataaatc atgtcgaaag ctacatataa ggaacgtgct gctactcatc
     1081 ctagtcctgt tgctgccaag ctatttaata tcatgcacga aaagcaaaca aacttgtgtg
     1141 cttcattgga tgttcgtacc accaaggaat tactggagtt agttgaagca ttaggtccca
     1201 aaatttgttt actaaaaaca catgtggata tcttgactga tttttccatg gagggcacag
     1261 ttaagccgct aaaggcatta tccgccaagt acaatttttt actcttcgaa gacagaaaat
     1321 ttgctgacat tggtaataca gtcaaattgc agtactctgc gggtgtatac agaatagcag
     1381 aatgggcaga cattacgaat gcacacggtg tggtgggccc aggtattgtt agcggtttga
     1441 agcaggcggc agaagaagta acaaaggaac ctagaggcct tttgatgtta gcagaattgt
     1501 catgcaaggg ctccctatct actggagaat atactaaggg tactgttgac attgcgaaga
     1561 gcgacaaaga ttttgttatc ggctttattg ctcaaagaga catgggtgga agagatgaag
     1621 gttacgattg gttgattatg acacccggtg tgggtttaga tgacaaggga gacgcattgg
     1681 gtcaacagta tagaaccgtg gatgatgtgg tctctacagg atctgacatt attattgttg
     1741 gaagaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag
     1801 caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact gtattataag
     1861 taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat cagttattac
     1921 cctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg
     1981 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     2041 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     2101 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2161 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2221 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     2281 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     2341 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     2401 ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg
     2461 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     2521 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     2581 ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct
     2641 cgagagcgac ctcatgctat acctgagaaa gcaacctgac ctacaggaaa gagttactca
     2701 agaataagaa ttttcgtttt aaaacctaag agtcacttta aaatttgtat acacttattt
     2761 tttttataac ttatttaata ataaaaatca taaatcataa gaaattcgct tatttagaag
     2821 tgtcaacaac gtatctacca acggaatgcg tgcgatgcac acaccatagc ttcaaaatgt
     2881 ttctactcct tttttactct tccagatttt ctcggactcc gcgcatcgcc gtaccacttc
     2941 aaaacaccca agcacagcat actaaatttc ccctctttct tcctctaggg tgtcgttaat
     3001 tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt ttcttcgtcg
     3061 aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg aaaatttttt tttttgattt
     3121 ttttctcttt cgatgacctc ccattgatat ttaagttaat aaacggtctt caatttctca
     3181 agtttcagtt tcatttttct tgttctatta caactttttt tacttcttgc tcattagaaa
     3241 gaaagcatag caatctaatc taagttttaa ttacaaatct gtcataaaac aatggacatg
     3301 ccgagaatca aacccggtca gagggttatg atggcgttgc gtaagatgat cgctagtggg
     3361 gaaatcaaat ccggcgaaag gattgcagag atcccaaccg cagccgcact gggggtttcc
     3421 aggatgccag tccgtacagc attgcgtagt ttggaacaag agggattggt ggttcgtttg
     3481 ggcgccagag gttacgcggc tcgtggtgtc agcagcgacc aaataagaga cgcgatcgaa
     3541 gttaggggag tactggaagg gttcgctgcc aggcgtttag ctgagcgtgg gatgacagcg
     3601 gaaacccatg cccgttttgt agctctaatt gctgagggag aggctttatt tgcagcaggg
     3661 cgtctaaacg gagaggatct tgacaggtat gctgcgtaca accaagcatt tcacgacacc
     3721 cttgtgtccg cggcaggcaa tggcgcggta gagtccgccc ttgcgcgtaa cggcttcgag
     3781 cccagggccg ctgctggagc attagcactt gacctaatgg acttgccagc ggagtatgaa
     3841 cacctgctgg cggcccacag acagcatcaa gccgtattgg atgcggtgtc atgtggcgat
     3901 gcagagggag cagagagaat catgagggac cacgctcttg cggcgatccg taacgcaaaa
     3961 gtttttgaag ctgctgcttc cgctggggct ccgctaggtg cggcctggag tataagagca
     4021 gactgataat cgcgtgcatt catccgctct aaccgaaaag gaaggagtta gacaacctga
     4081 agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt
     4141 tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac
     4201 cttgcttgag aagagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     4261 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4321 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4381 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4441 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4501 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4561 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4621 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4681 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4741 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4801 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4861 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4921 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4981 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5041 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5101 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5161 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5221 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5281 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5341 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5401 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5461 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5521 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5581 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5641 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5701 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5761 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5821 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5881 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5941 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6001 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6061 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6121 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6181 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6241 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6301 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6361 atgtatttag aaa
//
