LOCUS       sequence-467840-        7773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AvrSr62-5 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7773)
  AUTHORS   Chen R, Chen J, Outram MA, Powell OR, Arndell T, Gajendiran K, Wang 
            YL, Ayliffe MA, Blundell C, Figueroa M, Sperschneider J, Vanhercke 
            T, Tang D, Xu Y, Zhong G, Gardener C, Yu G, Gourdoupis S, Jaremko Ł,
            Matny O, Steffenson BJ, Boshoff WHP, Meyer WB, Arold ST, Dodds PN, 
            Wulff BBH
  TITLE     A wheat tandem kinase sensor activates an NLR helper to trigger 
            immunity
  JOURNAL   bioRxiv 2024.08.30.610287
REFERENCE   2  (bases 1 to 7773)
  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: "bioRxiv 
            2024.08.30.610287"
COMMENT     Sequence Label: pGBKT7_AvrSr62-5
FEATURES             Location/Qualifiers
     source          1..7773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        30..734
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             762..1202
                     /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     1213..1232
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1213..1231
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1251..1280
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     terminator      1808..1855
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1882..2069
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1937..1961)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2093..2109)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2093..2109)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2106..2128)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2117..2133
                     /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(2141..2147,2148..2165,2166..2171))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2186..2207
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2324..2341)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2495..3083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2575..2594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3412..3459
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3484..3503
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3691..4485)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3691..4485)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3802..3821)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4412..4431
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4486..4590)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4617..5959
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5580..5627)
                     /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     6000..6018
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6056..6078)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6178..6197
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6218..6499
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             6500..7174
                     /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(7277..7732)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7414..7435)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7626..7645
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cggtgcgggc ctcttcgcta ttacgccaga tccttttgtt gtttccgggt gtacaatatg
       61 gacttcctct tttctggcaa ccaaacccat acatcgggat tcctataata ccttcgttgg
      121 tctccctaac atgtaggtgg cggaggggag atatacaata gaacagatac cagacaagac
      181 ataatgggct aaacaagact acaccaatta cactgcctca ttgatggtgg tacataacga
      241 actaatactg tagccctaga cttgatagcc atcatcatat cgaagtttca ctaccctttt
      301 tccatttgcc atctattgaa gtaataatag gcgcatgcaa cttcttttct ttttttttct
      361 tttctctctc ccccgttgtt gtctcaccat atccgcaatg acaaaaaaat gatggaagac
      421 actaaaggaa aaaattaacg acaaagacag caccaacaga tgtcgttgtt ccagagctga
      481 tgaggggtat ctcgaagcac acgaaacttt ttccttcctt cattcacgca cactactctc
      541 taatgagcaa cggtatacgg ccttccttcc agttacttga atttgaaata aaaaaaagtt
      601 tgctgtcttg ctatcaagta taaatagacc tgcaattatt aatcttttgt ttcctcgtca
      661 ttgttctcgt tccctttctt ccttgtttct ttttctgcac aatatttcaa gctataccaa
      721 gcatacaatc aactccaagc ttgaagcaag cctcctgaaa gatgaagcta ctgtcttcta
      781 tcgaacaagc atgcgatatt tgccgactta aaaagctcaa gtgctccaaa gaaaaaccga
      841 agtgcgccaa gtgtctgaag aacaactggg agtgtcgcta ctctcccaaa accaaaaggt
      901 ctccgctgac tagggcacat ctgacagaag tggaatcaag gctagaaaga ctggaacagc
      961 tatttctact gatttttcct cgagaagacc ttgacatgat tttgaaaatg gattctttac
     1021 aggatataaa agcattgtta acaggattat ttgtacaaga taatgtgaat aaagatgccg
     1081 tcacagatag attggcttca gtggagactg atatgcctct aacattgaga cagcatagaa
     1141 taagtgcgac atcatcatcg gaagagagta gtaacaaagg tcaaagacag ttgactgtat
     1201 cgccggaatt tgtaatacga ctcactatag ggcgagccgc catcatggag gagcagaagc
     1261 tgatctcaga ggaggacctg catatggcca tggaggccga attcatgaag ccagtgatga
     1321 agcgctccat cgagaggttc ggcatggaag gctcctccag cgtggaagag tctccattcc
     1381 acgggccact ctacgagtcc aacgagtacc acaacatcaa cggcgacctc cacatcgtgt
     1441 cctggtactg cactccacca gagatcacct tccacttcaa cggcaaggac cgcaaggtga
     1501 cctaccacga gttcctcatg ctcggcgact ccgacggcat ctccaagtct gacaagcaag
     1561 ccatgaccaa cgcgctcatc agcaaggaac aaggctccaa ggtgatcggc atgtcccaaa
     1621 tcggcaacca gccaatctcc gccgaagaga agaaccgcaa gtggccaaat ggcccaccac
     1681 ggttcatcaa gaccaccgac atgatccact ccaacaccga cggccgcaag atcgtgtacc
     1741 gctacttcag gaccactcca gtggccgaga agatctgagg atccgtcgac ctgcagcggc
     1801 cgcataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgcgcgc
     1861 ttgcagccaa gctaattccg ggcgaatttc ttatgattta tgatttttat tattaaataa
     1921 gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa
     1981 ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagcatgag
     2041 gtcgctctta ttgaccacac ctctaccggc atgcaagctt ggcgtaatca tggtcatagc
     2101 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     2161 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     2221 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2281 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2341 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2401 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2461 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2521 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2581 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2641 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2701 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2761 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2821 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2881 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     2941 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3001 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3061 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3121 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3181 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaac
     3241 ctgaggctat ggcagggcct gccgccccga cgttggctgc gagccctggg ccttcacccg
     3301 aacttggggg gtggggtggg gaaaaggaag aaacgcgggc gtattggccc caatggggtc
     3361 tcggtggggt atcgacagag tgccagccct gggaccgaac cccgcgttta tgaacaaacg
     3421 acccaacacc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg gttccttccg
     3481 gtattgtctc cttccgtgtt tcagttagcc tccccctagg gtgggcgaag aactccagca
     3541 tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca
     3601 acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt
     3661 ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa
     3721 ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
     3781 ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc
     3841 cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat
     3901 attcggcaag caggcatcgc catgagtcac gacgagatcc tcgccgtcgg gcatgctcgc
     3961 cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc
     4021 ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
     4081 gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat
     4141 gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc
     4201 gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg
     4261 aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcttgcagtt cattcagggc
     4321 accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
     4381 ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac
     4441 ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatactct tcctttttca
     4501 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4561 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaacg
     4621 aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa
     4681 caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg ctattttacc
     4741 aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt
     4801 tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga gagcgctatt
     4861 ttaccaacaa agaatctata cttctttttt gttctacaaa aatgcatccc gagagcgcta
     4921 tttttctaac aaagcatctt agattacttt ttttctcctt tgtgcgctct ataatgcagt
     4981 ctcttgataa ctttttgcac tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc
     5041 tattttctct tccataaaaa aagcctgact ccacttcccg cgtttactga ttactagcga
     5101 agctgcgggt gcattttttc aagataaagg catccccgat tatattctat accgatgtgg
     5161 attgcgcata ctttgtgaac agaaagtgat agcgttgatg attcttcatt ggtcagaaaa
     5221 ttatgaacgg tttcttctat tttgtctcta tatactacgt ataggaaatg tttacatttt
     5281 cgtattgttt tcgattcact ctatgaatag ttcttactac aatttttttg tctaaagagt
     5341 aatactagag ataaacataa aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg
     5401 aaaggtggat gggtaggtta tatagggata tagcacagag atatatagca aagagatact
     5461 tttgagcaat gtttgtggaa gcggtattcg caatatttta gtagctcgtt acagtccggt
     5521 gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg
     5581 aagttcctat actttctaga gaataggaac ttcggaatag gaacttcaaa gcgtttccga
     5641 aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat acagctcact gttcacgtcg
     5701 cacctatatc tgcgtgttgc ctgtatatat atatacatga gaagaacggc atagtgcgtg
     5761 tttatgctta aatgcgtact tatatgcgtc tatttatgta ggatgaaagg tagtctagta
     5821 cctcctgtga tattatccca ttccatgcgg ggtatcgtat gcttccttca gcactaccct
     5881 ttagctgttc tatatgctgc cactcctcaa ttggattagt ctcatccttc aatgctatca
     5941 tttcctttga tattggatca tactaagaaa ccattattat catgacatta acctataaaa
     6001 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     6061 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     6121 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     6181 catcagagca gattgtactg agagtgcacc atagatcaac gacattacta tatatataat
     6241 ataggaagca tttaatagaa cagcatcgta atatatgtgt actttgcagt tatgacgcca
     6301 gatggcagta gtggaagata ttctttattg aaaaatagct tgtcacctta cgtacaatct
     6361 tgatccggag cttttctttt tttgccgatt aagaattaat tcggtcgaaa aaagaaaagg
     6421 agagggccaa gagggagggc attggtgact attgagcacg tgagtatacg tgattaagca
     6481 cacaaaggca gcttggagta tgtctgttat taatttcaca ggtagttctg gtccattggt
     6541 gaaagtttgc ggcttgcaga gcacagaggc cgcagaatgt gctctagatt ccgatgctga
     6601 cttgctgggt attatatgtg tgcccaatag aaagagaaca attgacccgg ttattgcaag
     6661 gaaaatttca agtcttgtaa aagcatataa aaatagttca ggcactccga aatacttggt
     6721 tggcgtgttt cgtaatcaac ctaaggagga tgttttggct ctggtcaatg attacggcat
     6781 tgatatcgtc caactgcatg gagatgagtc gtggcaagaa taccaagagt tcctcggttt
     6841 gccagttatt aaaagactcg tatttccaaa agactgcaac atactactca gtgcagcttc
     6901 acagaaacct cattcgttta ttcccttgtt tgattcagaa gcaggtggga caggtgaact
     6961 tttggattgg aactcgattt ctgactgggt tggaaggcaa gagagccccg aaagcttaca
     7021 ttttatgtta gctggtggac tgacgccaga aaatgttggt gatgcgctta gattaaatgg
     7081 cgttattggt gttgatgtaa gcggaggtgt ggagacaaat ggtgtaaaag actctaacaa
     7141 aatagcaaat ttcgtcaaaa atgctaagaa ataggttatt actgagtagt atttatttaa
     7201 gtattgtttg tgcacttgcc gatctatgcg gtgtgaaata ccgcacagat gcgtaaggag
     7261 aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     7321 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     7381 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     7441 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     7501 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     7561 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     7621 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     7681 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc
     7741 cattcaggct gcgcaactgt tgggaagggc gat
//
