LOCUS       sequence-471095-        7577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast two-hybrid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7577)
  AUTHORS   Sung YC, Li Y, Bernasconi Z, Baik S, Asuke S, Keller B, Fahima T, 
            Coaker G
  TITLE     Wheat tandem kinase RWT4 directly binds a fungal effector to 
            activate defense.
  JOURNAL   Nat Genet. 2025 Apr 14. doi: 10.1038/s41588-025-02162-w.
  PUBMED    40229601
REFERENCE   2  (bases 1 to 7577)
  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: "Nat Genet. 
            2025 Apr 14. doi: 10.1038/s41588-025-02162-w."
COMMENT     Sequence Label: pGBKT7-AvrPWT4
FEATURES             Location/Qualifiers
     source          1..7577
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    329..376
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     401..420
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(608..1402)
                     /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(608..1402)
                     /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(719..738)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1329..1348
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1403..1507)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1534..2876
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(2497..2544)
                     /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     2917..2935
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2973..2995)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3095..3114
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3135..3416
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             3417..4091
                     /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(4194..4649)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4331..4352)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4543..4562
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4720..5424
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             5452..5892
                     /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     5903..5922
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5903..5921
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5941..5970
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     protein_bind    5980..6004
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(6244..6268)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      6302..6349
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      6376..6563
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(6431..6455)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(6587..6603)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6587..6603)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6600..6622)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6611..6627
                     /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(6635..6641,6642..6659,6660..6665))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6680..6701
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6818..6835)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6989..7577)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7069..7088)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaacctg aggctatggc agggcctgcc
      181 gccccgacgt tggctgcgag ccctgggcct tcacccgaac ttggggggtg gggtggggaa
      241 aaggaagaaa cgcgggcgta ttggccccaa tggggtctcg gtggggtatc gacagagtgc
      301 cagccctggg accgaacccc gcgtttatga acaaacgacc caacaccgtg cgttttattc
      361 tgtcttttta ttgccgtcat agcgcgggtt ccttccggta ttgtctcctt ccgtgtttca
      421 gttagcctcc ccctagggtg ggcgaagaac tccagcatga gatccccgcg ctggaggatc
      481 atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa ggcggcggtg
      541 gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc gaaccccaga
      601 gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
      661 cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
      721 tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt
      781 cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat
      841 gagtcacgac gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg
      901 ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
      961 tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
     1021 gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
     1081 caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
     1141 ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
     1201 atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa
     1261 aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
     1321 tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
     1381 gcaatccatc ttgttcaatc atactcttcc tttttcaata ttattgaagc atttatcagg
     1441 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     1501 ttccgcgcac atttccccga aaagtgccac ctgaacgaag catctgtgct tcattttgta
     1561 gaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt
     1621 acagaacaga aatgcaacgc gaaagcgcta ttttaccaac gaagaatctg tgcttcattt
     1681 ttgtaaaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca
     1741 tttttacaga acagaaatgc aacgcgagag cgctatttta ccaacaaaga atctatactt
     1801 cttttttgtt ctacaaaaat gcatcccgag agcgctattt ttctaacaaa gcatcttaga
     1861 ttactttttt tctcctttgt gcgctctata atgcagtctc ttgataactt tttgcactgt
     1921 aggtccgtta aggttagaag aaggctactt tggtgtctat tttctcttcc ataaaaaaag
     1981 cctgactcca cttcccgcgt ttactgatta ctagcgaagc tgcgggtgca ttttttcaag
     2041 ataaaggcat ccccgattat attctatacc gatgtggatt gcgcatactt tgtgaacaga
     2101 aagtgatagc gttgatgatt cttcattggt cagaaaatta tgaacggttt cttctatttt
     2161 gtctctatat actacgtata ggaaatgttt acattttcgt attgttttcg attcactcta
     2221 tgaatagttc ttactacaat ttttttgtct aaagagtaat actagagata aacataaaaa
     2281 atgtagaggt cgagtttaga tgcaagttca aggagcgaaa ggtggatggg taggttatat
     2341 agggatatag cacagagata tatagcaaag agatactttt gagcaatgtt tgtggaagcg
     2401 gtattcgcaa tattttagta gctcgttaca gtccggtgcg tttttggttt tttgaaagtg
     2461 cgtcttcaga gcgcttttgg ttttcaaaag cgctctgaag ttcctatact ttctagagaa
     2521 taggaacttc ggaataggaa cttcaaagcg tttccgaaaa cgagcgcttc cgaaaatgca
     2581 acgcgagctg cgcacataca gctcactgtt cacgtcgcac ctatatctgc gtgttgcctg
     2641 tatatatata tacatgagaa gaacggcata gtgcgtgttt atgcttaaat gcgtacttat
     2701 atgcgtctat ttatgtagga tgaaaggtag tctagtacct cctgtgatat tatcccattc
     2761 catgcggggt atcgtatgct tccttcagca ctacccttta gctgttctat atgctgccac
     2821 tcctcaattg gattagtctc atccttcaat gctatcattt cctttgatat tggatcatac
     2881 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     2941 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     3001 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     3061 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     3121 gtgcaccata gatcaacgac attactatat atataatata ggaagcattt aatagaacag
     3181 catcgtaata tatgtgtact ttgcagttat gacgccagat ggcagtagtg gaagatattc
     3241 tttattgaaa aatagcttgt caccttacgt acaatcttga tccggagctt ttcttttttt
     3301 gccgattaag aattaattcg gtcgaaaaaa gaaaaggaga gggccaagag ggagggcatt
     3361 ggtgactatt gagcacgtga gtatacgtga ttaagcacac aaaggcagct tggagtatgt
     3421 ctgttattaa tttcacaggt agttctggtc cattggtgaa agtttgcggc ttgcagagca
     3481 cagaggccgc agaatgtgct ctagattccg atgctgactt gctgggtatt atatgtgtgc
     3541 ccaatagaaa gagaacaatt gacccggtta ttgcaaggaa aatttcaagt cttgtaaaag
     3601 catataaaaa tagttcaggc actccgaaat acttggttgg cgtgtttcgt aatcaaccta
     3661 aggaggatgt tttggctctg gtcaatgatt acggcattga tatcgtccaa ctgcatggag
     3721 atgagtcgtg gcaagaatac caagagttcc tcggtttgcc agttattaaa agactcgtat
     3781 ttccaaaaga ctgcaacata ctactcagtg cagcttcaca gaaacctcat tcgtttattc
     3841 ccttgtttga ttcagaagca ggtgggacag gtgaactttt ggattggaac tcgatttctg
     3901 actgggttgg aaggcaagag agccccgaaa gcttacattt tatgttagct ggtggactga
     3961 cgccagaaaa tgttggtgat gcgcttagat taaatggcgt tattggtgtt gatgtaagcg
     4021 gaggtgtgga gacaaatggt gtaaaagact ctaacaaaat agcaaatttc gtcaaaaatg
     4081 ctaagaaata ggttattact gagtagtatt tatttaagta ttgtttgtgc acttgccgat
     4141 ctatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt
     4201 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     4261 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     4321 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     4381 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     4441 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     4501 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     4561 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     4621 cgcgcttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg
     4681 gaagggcgat cggtgcgggc ctcttcgcta ttacgccaga tccttttgtt gtttccgggt
     4741 gtacaatatg gacttcctct tttctggcaa ccaaacccat acatcgggat tcctataata
     4801 ccttcgttgg tctccctaac atgtaggtgg cggaggggag atatacaata gaacagatac
     4861 cagacaagac ataatgggct aaacaagact acaccaatta cactgcctca ttgatggtgg
     4921 tacataacga actaatactg tagccctaga cttgatagcc atcatcatat cgaagtttca
     4981 ctaccctttt tccatttgcc atctattgaa gtaataatag gcgcatgcaa cttcttttct
     5041 ttttttttct tttctctctc ccccgttgtt gtctcaccat atccgcaatg acaaaaaaat
     5101 gatggaagac actaaaggaa aaaattaacg acaaagacag caccaacaga tgtcgttgtt
     5161 ccagagctga tgaggggtat ctcgaagcac acgaaacttt ttccttcctt cattcacgca
     5221 cactactctc taatgagcaa cggtatacgg ccttccttcc agttacttga atttgaaata
     5281 aaaaaaagtt tgctgtcttg ctatcaagta taaatagacc tgcaattatt aatcttttgt
     5341 ttcctcgtca ttgttctcgt tccctttctt ccttgtttct ttttctgcac aatatttcaa
     5401 gctataccaa gcatacaatc aactccaagc ttgaagcaag cctcctgaaa gatgaagcta
     5461 ctgtcttcta tcgaacaagc atgcgatatt tgccgactta aaaagctcaa gtgctccaaa
     5521 gaaaaaccga agtgcgccaa gtgtctgaag aacaactggg agtgtcgcta ctctcccaaa
     5581 accaaaaggt ctccgctgac tagggcacat ctgacagaag tggaatcaag gctagaaaga
     5641 ctggaacagc tatttctact gatttttcct cgagaagacc ttgacatgat tttgaaaatg
     5701 gattctttac aggatataaa agcattgtta acaggattat ttgtacaaga taatgtgaat
     5761 aaagatgccg tcacagatag attggcttca gtggagactg atatgcctct aacattgaga
     5821 cagcatagaa taagtgcgac atcatcatcg gaagagagta gtaacaaagg tcaaagacag
     5881 ttgactgtat cgccggaatt tgtaatacga ctcactatag ggcgagccgc catcatggag
     5941 gagcagaagc tgatctcaga ggaggacctg cataatcaaa caagtttgta caaaaaagca
     6001 ggctccacca tgaaaccccc ctccgattcg acggatgcct caaaaatccc gacttacaaa
     6061 attaaacatc cagggcctcg ttacattata actgacgatg gtcgagttgt tgaggtcgaa
     6121 cgcaaggaat caggacgcta tcaattaatc gatggggttt tagtacgtgt ggaatatgaa
     6181 aagaaaaccg ggggttcgga agggtcaaaa acatccacca acacgcgcgg tccgaaaatc
     6241 tagacccagc tttcttgtac aaagtggttc gatgatccgt cgacctgcag cggccgcata
     6301 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc gcgcttgcag
     6361 ccaagctaat tccgggcgaa tttcttatga tttatgattt ttattattaa ataagttata
     6421 aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta
     6481 ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca tgaggtcgct
     6541 cttattgacc acacctctac cggcatgcaa gcttggcgta atcatggtca tagctgtttc
     6601 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     6661 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     6721 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6781 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6841 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6901 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6961 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     7021 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     7081 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     7141 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     7201 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     7261 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     7321 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     7381 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     7441 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     7501 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     7561 gaaaaaaagg atctcaa
//
