LOCUS       sequence-319279-        6591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Wild type VanR where the residue in position 165 was mutated from 
            Phenylalanine to Valine. 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 6591)
  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 6591)
  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 - F165V
FEATURES             Location/Qualifiers
     source          1..6591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..65
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        169..575
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     complement(1542..1562)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1542..1560)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1581..1597)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1581..1597)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1594..1616)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1605..1621
                     /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(1629..1635,1636..1653,1654..1659))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1674..1695
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1812..1829)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1983..2571)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2063..2082)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2742..3533,3534..3602))
                     /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     3365..3384
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3603..3707)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(3821..3925)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    3962..4465
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     4506..4524
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4562..4584)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4684..4703
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4727..4942
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             4943..5746
                     /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(5877..6332)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6014..6035)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6226..6245
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6458..6480
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6472..6489
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6473..6489
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6499..6518
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6499..6517
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(join(6546..6591,1..120))
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
ORIGIN
        1 agagttactc aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta
       61 tacacttatt ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc
      121 ttatttagaa gtgtcaacaa cgtatctacc aacggaatgc gtgcgatgca cacaccatag
      181 cttcaaaatg tttctactcc ttttttactc ttccagattt tctcggactc cgcgcatcgc
      241 cgtaccactt caaaacaccc aagcacagca tactaaattt cccctctttc ttcctctagg
      301 gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc ctcgtttctt
      361 tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt tctttttctt gaaaattttt
      421 ttttttgatt tttttctctt tcgatgacct cccattgata tttaagttaa taaacggtct
      481 tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg
      541 ctcattagaa agaaagcata gcaatctaat ctaagtttta attacaaatc tgtcataaaa
      601 caatggacat gccgagaatc aaacccggtc agagggttat gatggcgttg cgtaagatga
      661 tcgctagtgg ggaaatcaaa tccggcgaaa ggattgcaga gatcccaacc gcagccgcac
      721 tgggggtttc caggatgcca gtccgtacag cattgcgtag tttggaacaa gagggattgg
      781 tggttcgttt gggcgccaga ggttacgcgg ctcgtggtgt cagcagcgac caaataagag
      841 acgcgatcga agttagggga gtactggaag ggttcgctgc caggcgttta gctgagcgtg
      901 ggatgacagc ggaaacccat gcccgttttg tagctctaat tgctgaggga gaggctttat
      961 ttgcagcagg gcgtctaaac ggagaggatc ttgacaggta tgctgcgtac aaccaagcat
     1021 ttcacgacac ccttgtgtcc gcggcaggca atggcgcggt agagtccgcc cttgcgcgta
     1081 acggcttcga gcccgtggcc gctgctggag cattagcact tgacctaatg gacttgccag
     1141 cggagtatga acacctgctg gcggcccaca gacagcatca agccgtattg gatgcggtgt
     1201 catgtggcga tgcagaggga gcagagagaa tcatgaggga ccacgctctt gcggcgatcc
     1261 gtaacgcaaa agtttttgaa gctgctgctt ccgctggggc tccgctaggt gcggcctgga
     1321 gtataagagc agactgataa tcgcgtgcat tcatccgctc taaccgaaaa ggaaggagtt
     1381 agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg
     1441 ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt
     1501 atactgaaaa ccttgcttga gaagagctcc agcttttgtt ccctttagtg agggttaatt
     1561 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1621 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1681 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1741 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1801 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1861 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1921 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1981 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2041 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2101 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2161 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2221 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2281 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2341 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2401 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2461 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2521 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2581 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2641 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2701 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2761 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2821 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2881 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2941 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3001 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3061 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3121 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3181 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3241 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3301 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3361 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3421 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3481 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3541 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3601 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3661 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3721 aaagtgccac ctgggtcctt ttcatcgggt gagcaaaaac aggaaggcaa aatgccgcaa
     3781 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     3841 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     3901 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggtcctttt
     3961 catcacgtgc tataaaaata attataattt aaatttttta atataaatat ataaattaaa
     4021 aatagaaagt aaaaaaagaa attaaagaaa aaatagtttt tgttttccga agatgtaaaa
     4081 gactctaggg ggatcgccaa caaatactac cttttatctt gctcttcctg ctctcaggta
     4141 ttaatgccga attgtttcat cttgtctgtg tagaagacca cacacgaaaa tcctgtgatt
     4201 ttacatttta cttatcgtta atcgaatgta tatctattta atctgctttt cttgtctaat
     4261 aaatatatat gtaaagtacg ctttttgttg aaatttttta aacctttgtt tatttttttt
     4321 tcttcattcc gtaactcttc taccttcttt atttactttc taaaatccaa atacaaaaca
     4381 taaaaataaa taaacacaga gtaaattccc aaattattcc atcattaaaa gatacgaggc
     4441 gcgtgtaagt tacaggcaag cgatccgtct aagaaaccat tattatcatg acattaacct
     4501 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     4561 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     4621 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     4681 atgcggcatc agagcagatt gtactgagag tgcaccatac caccttttca attcatcatt
     4741 ttttttttat tctttttttt gatttcggtt tccttgaaat ttttttgatt cggtaatctc
     4801 cgaacagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata tacgcatatg
     4861 tagtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca gaacaaaaac
     4921 ctgcaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg ctgctactca
     4981 tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa caaacttgtg
     5041 tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag cattaggtcc
     5101 caaaatttgt ttactaaaaa cacatgtgga tatcttgact gatttttcca tggagggcac
     5161 agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg aagacagaaa
     5221 atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat acagaatagc
     5281 agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg ttagcggttt
     5341 gaagcaggcg gcagaagaag taacaaagga acctagaggc cttttgatgt tagcagaatt
     5401 gtcatgcaag ggctccctat ctactggaga atatactaag ggtactgttg acattgcgaa
     5461 gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg gaagagatga
     5521 aggttacgat tggttgatta tgacacccgg tgtgggttta gatgacaagg gagacgcatt
     5581 gggtcaacag tatagaaccg tggatgatgt ggtctctaca ggatctgaca ttattattgt
     5641 tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac gttacagaaa
     5701 agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaaaaaa ctgtattata
     5761 agtaaatgca tgtatactaa actcacaaat tagagcttca atttaattat atcagttatt
     5821 accctatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat
     5881 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     5941 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     6001 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     6061 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     6121 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     6181 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     6241 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     6301 cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt
     6361 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     6421 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     6481 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc
     6541 ctcgagagcg acctcatgct atacctgaga aagcaacctg acctacagga a
//
