LOCUS       sequence-467839-        7770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AvrSr62-1 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7770)
  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 7770)
  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_AvrSr62-1
FEATURES             Location/Qualifiers
     source          1..7770
                     /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      1805..1852
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1879..2066
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1934..1958)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2090..2106)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2090..2106)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2103..2125)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2114..2130
                     /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(2138..2144,2145..2162,2163..2168))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2183..2204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2321..2338)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2492..3080)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2572..2591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3409..3456
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3481..3500
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3688..4482)
                     /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(3688..4482)
                     /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(3799..3818)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4409..4428
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4483..4587)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4614..5956
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5577..5624)
                     /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     5997..6015
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6053..6075)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6175..6194
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6215..6496
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             6497..7171
                     /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(7274..7729)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7411..7432)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7623..7642
                     /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 ccaatcatga
     1321 agcgctccat cgagaggttc ggcatggaag gctcctccag cgtggaagag tctccattct
     1381 acggcccact ctacgagtcc aaggaatact cctccgccaa cggcaagctc cacaccgtgt
     1441 cttggtactt caccccacca gagatcacct tccacttcaa cgagaaggac cgcaaggtgc
     1501 cataccacga gttcctcaag ctcggcgact ccgacggcat ctccaagtct gagaaggacg
     1561 agatcaccgc gctcctcagc cgcaatcaag gctctaaggt gatcggcatg tccaagatca
     1621 ccaaccagcc aatctccgcc gacgagaaga accggaagtg gccaaacgga ccaccacgct
     1681 tcatcaaggc caccgacatg ctccactcta agaccgacgg caagaggatc gtgtacaagt
     1741 acttccacac taacccagtg gtcgagaaga tgtgaggatc cgtcgacctg cagcggccgc
     1801 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgcgcgcttg
     1861 cagccaagct aattccgggc gaatttctta tgatttatga tttttattat taaataagtt
     1921 ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa acgaaaattc
     1981 ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata gcatgaggtc
     2041 gctcttattg accacacctc taccggcatg caagcttggc gtaatcatgg tcatagctgt
     2101 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     2161 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     2221 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2281 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2341 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2401 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2461 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2521 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2581 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2641 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2701 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2761 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2821 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2881 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2941 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3001 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3061 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3121 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3181 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaacctg
     3241 aggctatggc agggcctgcc gccccgacgt tggctgcgag ccctgggcct tcacccgaac
     3301 ttggggggtg gggtggggaa aaggaagaaa cgcgggcgta ttggccccaa tggggtctcg
     3361 gtggggtatc gacagagtgc cagccctggg accgaacccc gcgtttatga acaaacgacc
     3421 caacaccgtg cgttttattc tgtcttttta ttgccgtcat agcgcgggtt ccttccggta
     3481 ttgtctcctt ccgtgtttca gttagcctcc ccctagggtg ggcgaagaac tccagcatga
     3541 gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc
     3601 tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt
     3661 cggtcatttc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc
     3721 gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
     3781 gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
     3841 cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
     3901 cggcaagcag gcatcgccat gagtcacgac gagatcctcg ccgtcgggca tgctcgcctt
     3961 gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
     4021 atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
     4081 gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
     4141 ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
     4201 caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
     4261 gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc
     4321 ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
     4381 ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
     4441 agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atactcttcc tttttcaata
     4501 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4561 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgaacgaag
     4621 catctgtgct tcattttgta gaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa
     4681 agaatctgag ctgcattttt acagaacaga aatgcaacgc gaaagcgcta ttttaccaac
     4741 gaagaatctg tgcttcattt ttgtaaaaca aaaatgcaac gcgagagcgc taatttttca
     4801 aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgagag cgctatttta
     4861 ccaacaaaga atctatactt cttttttgtt ctacaaaaat gcatcccgag agcgctattt
     4921 ttctaacaaa gcatcttaga ttactttttt tctcctttgt gcgctctata atgcagtctc
     4981 ttgataactt tttgcactgt aggtccgtta aggttagaag aaggctactt tggtgtctat
     5041 tttctcttcc ataaaaaaag cctgactcca cttcccgcgt ttactgatta ctagcgaagc
     5101 tgcgggtgca ttttttcaag ataaaggcat ccccgattat attctatacc gatgtggatt
     5161 gcgcatactt tgtgaacaga aagtgatagc gttgatgatt cttcattggt cagaaaatta
     5221 tgaacggttt cttctatttt gtctctatat actacgtata ggaaatgttt acattttcgt
     5281 attgttttcg attcactcta tgaatagttc ttactacaat ttttttgtct aaagagtaat
     5341 actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca aggagcgaaa
     5401 ggtggatggg taggttatat agggatatag cacagagata tatagcaaag agatactttt
     5461 gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca gtccggtgcg
     5521 tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag cgctctgaag
     5581 ttcctatact ttctagagaa taggaacttc ggaataggaa cttcaaagcg tttccgaaaa
     5641 cgagcgcttc cgaaaatgca acgcgagctg cgcacataca gctcactgtt cacgtcgcac
     5701 ctatatctgc gtgttgcctg tatatatata tacatgagaa gaacggcata gtgcgtgttt
     5761 atgcttaaat gcgtacttat atgcgtctat ttatgtagga tgaaaggtag tctagtacct
     5821 cctgtgatat tatcccattc catgcggggt atcgtatgct tccttcagca ctacccttta
     5881 gctgttctat atgctgccac tcctcaattg gattagtctc atccttcaat gctatcattt
     5941 cctttgatat tggatcatac taagaaacca ttattatcat gacattaacc tataaaaata
     6001 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     6061 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     6121 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     6181 cagagcagat tgtactgaga gtgcaccata gatcaacgac attactatat atataatata
     6241 ggaagcattt aatagaacag catcgtaata tatgtgtact ttgcagttat gacgccagat
     6301 ggcagtagtg gaagatattc tttattgaaa aatagcttgt caccttacgt acaatcttga
     6361 tccggagctt ttcttttttt gccgattaag aattaattcg gtcgaaaaaa gaaaaggaga
     6421 gggccaagag ggagggcatt ggtgactatt gagcacgtga gtatacgtga ttaagcacac
     6481 aaaggcagct tggagtatgt ctgttattaa tttcacaggt agttctggtc cattggtgaa
     6541 agtttgcggc ttgcagagca cagaggccgc agaatgtgct ctagattccg atgctgactt
     6601 gctgggtatt atatgtgtgc ccaatagaaa gagaacaatt gacccggtta ttgcaaggaa
     6661 aatttcaagt cttgtaaaag catataaaaa tagttcaggc actccgaaat acttggttgg
     6721 cgtgtttcgt aatcaaccta aggaggatgt tttggctctg gtcaatgatt acggcattga
     6781 tatcgtccaa ctgcatggag atgagtcgtg gcaagaatac caagagttcc tcggtttgcc
     6841 agttattaaa agactcgtat ttccaaaaga ctgcaacata ctactcagtg cagcttcaca
     6901 gaaacctcat tcgtttattc ccttgtttga ttcagaagca ggtgggacag gtgaactttt
     6961 ggattggaac tcgatttctg actgggttgg aaggcaagag agccccgaaa gcttacattt
     7021 tatgttagct ggtggactga cgccagaaaa tgttggtgat gcgcttagat taaatggcgt
     7081 tattggtgtt gatgtaagcg gaggtgtgga gacaaatggt gtaaaagact ctaacaaaat
     7141 agcaaatttc gtcaaaaatg ctaagaaata ggttattact gagtagtatt tatttaagta
     7201 ttgtttgtgc acttgccgat ctatgcggtg tgaaataccg cacagatgcg taaggagaaa
     7261 ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     7321 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     7381 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     7441 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     7501 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     7561 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     7621 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     7681 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc cattcgccat
     7741 tcaggctgcg caactgttgg gaagggcgat
//
