LOCUS       sequence-478720-        8125 bp    DNA     circular SYN 15-JUL-2025
DEFINITION  Protoplast expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8125)
  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 8125)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 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: pUC19::ZmUBI-RWT4Ca_K217R/K327R
FEATURES             Location/Qualifiers
     source          1..8125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    3010..3034
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5956..6021
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(6028..6052)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6081..6170
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      6204..6456
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(6500..6517)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6500..6516)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6509..6531)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6725..6744)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6844..6866
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6904..6922)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6990..7094
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7095..7163,7164..7955)
                     /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     complement(7313..7332)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttt gcagcgtgac
     1021 ccggtcgtgc ccctctctag agataatgag cattgcatgt ctaagttata aaaaattacc
     1081 acatattttt tttgtcacac ttgtttgaag tgcagtttat ctatctttat acatatattt
     1141 aaactttact ctacgaataa tataatctat agtactacaa taatatcagt gttttagaga
     1201 atcatataaa tgaacagtta gacatggtct aaaggacaat tgagtatttt gacaacagga
     1261 ctctacagtt ttatcttttt agtgtgcatg tgttctcctt tttttttgca aatagcttca
     1321 cctatataat acttcatcca ttttattagt acatccattt agggtttagg gttaatggtt
     1381 tttatagact aattttttta gtacatctat tttattctat tttagcctct aaattaagaa
     1441 aactaaaact ctattttagt ttttttattt aataatttag atataaaata gaataaaata
     1501 aagtgactaa aaattaaaca aatacccttt aagaaattaa aaaaactaag gaaacatttt
     1561 tcttgtttcg agtagataat gccagcctgt taaacgccgt cgacgagtct aacggacacc
     1621 aaccagcgaa ccagcagcgt cgcgtcgggc caagcgaagc agacggcacg gcatctctgt
     1681 cgctgcctct ggacccctct cgagagttcc gctccaccgt tggacttgct ccgctgtcgg
     1741 catccagaaa ttgcgtggcg gagcggcaga cgtgagccgg cacggcaggc ggcctcctcc
     1801 tcctctcacg gcaccggcag ctacggggga ttcctttccc accgctcctt cgctttccct
     1861 tcctcgcccg ccgtaataaa tagacacccc ctccacaccc tctttcccca acctcgtgtt
     1921 gttcggagcg cacacacaca caaccagatc tcccccaaat ccacccgtcg gcacctccgc
     1981 ttcaaggtac gccgctcgtc ctcccccccc ccccctctct accttctcta gatcggcgtt
     2041 ccggtccatg gttagggccc ggtagttcta cttctgttca tgtttgtgtt agatccgtgt
     2101 ttgtgttaga tccgtgctgc tagcgttcgt acacggatgc gacctgtacg tcagacacgt
     2161 tctgattgct aacttgccag tgtttctctt tggggaatcc tgggatggct ctagccgttc
     2221 cgcagacggg atcgatttca tgattttttt tgtttcgttg catagggttt ggtttgccct
     2281 tttcctttat ttcaatatat gccgtgcact tgtttgtcgg gtcatctttt catgcttttt
     2341 tttgtcttgg ttgtgatgat gtggtctggt tgggcggtcg ttctagatcg gagtagaatt
     2401 ctgtttcaaa ctacctggtg gatttattaa ttttggatct gtatgtgtgt gccatacata
     2461 ttcatagtta cgaattgaag atgatggatg gaaatatcga tctaggatag gtatacatgt
     2521 tgatgcgggt tttactgatg catatacaga gatgcttttt gttcgcttgg ttgtgatgat
     2581 gtggtgtggt tgggcggtcg ttcattcgtt ctagatcgga gtagaatact gtttcaaact
     2641 acctggtgta tttattaatt ttggaactgt atgtgtgtgt catacatctt catagttacg
     2701 agtttaagat ggatggaaat atcgatctag gataggtata catgttgatg tgggttttac
     2761 tgatgcatat acatgatggc atatgcagca tctattcata tgctctaacc ttgagtacct
     2821 atctattata ataaacaagt atgttttata attattttga tcttgatata cttggatgat
     2881 ggcatatgca gcagctatat gtggattttt ttagccctgc cttcatacgc tatttatttg
     2941 cttggtactg tttcttttgt cgatgctcac cctgttgttt ggtgttactt ctgcagacta
     3001 gagttatcaa caagtttgta caaaaaagca ggctccacca tgatgggtgg ctatgagttc
     3061 cagagggcag aattggacgc ccttgagggt gtagttcgtg acccaaccgc cgaacccatg
     3121 tctttaacac tacctctttt gcgtcacatt acgaatgatt tctcccctga gttcgagata
     3181 tctaaggacg actccgccgt ggtatatcta ggagtgctac catcaggatt tagggtggca
     3241 gtgaagaaat cacattttag gttttgtctg gacgacgagg acgccttcac taatgaagtt
     3301 tcaattgcaa tgaaagccgc acataagaat actgtaagag taatcggcta ttgtcatcat
     3361 acacatgagc agattgccga gtatgaggga aagcaagtct tcgcagaagt gcgtgagagg
     3421 ttgatatgta ctgagtacgt gcccaatgga cctctatccg gccatatcga aggcaaaatt
     3481 tgtgcacaaa tggacggcta cgagttccag cgtgctgagc ttgatgcatt ggagagagtt
     3541 gttagagata cctctgccga gccaatgtcc ttgacactgc cccttttgcg tcatatcaca
     3601 aatgatttct cagatgaatc caggatcgga cgtggaggat tcgctgttgt ctatcttggt
     3661 gttttgccat ccggcctgag aatcgccgtt agaagactgt caaatatcgc atatatgaat
     3721 gaaagtgcat tccagaacga agtattcatc acaatgaaag ccacacataa aaatacagtg
     3781 agattcatgg gatactgctc acagattcaa ggcaaactaa tagaacatga tggacaacat
     3841 gtctttgcac agcttgaaga gaggctaatt tgtgtggaat acgcacctaa gggaacatta
     3901 gacgctcaca tcggagatta tggtgaactg gactggaacc aaagatatca gattctgaaa
     3961 ggcatatgtc agggtttaca tcatctacat gacgaaatgc atgtctttca tggagatatc
     4021 cgccccgcca acattggaga tatccgcccc gccaacattc agatatccgc cccgccaaca
     4081 tggagatatc cgccccgcca acattcggag atatccgccc cgccaacatt tggagatatc
     4141 cgccccgcca acagagatat ccgccccgcc aacattggag atatccgccc cgccaacatt
     4201 cttatcggcg ataacctagt tcctaaaatc tatgatttcg gattgagtca aatgttcgag
     4261 gaggaggaaa cggaaagaat tgtggaaaac attgccggta cattcggcta catggcacct
     4321 gagttctgta caaacaacat ggtatccttc aaggccgaga tttacagttt aggcgttgta
     4381 ataggagaac ttcttatcgg taaaaaagga tggtttgacg aggacgtgcg gaagcttttt
     4441 gttcagcagt tgaaaggcct tcgcaaaact ttggtgaagg aaggcgcgtt ttccagctgg
     4501 gagaataaat accatcaagt gcgcacgtgt atggagattg gtcaggattg catcgatcca
     4561 aatcctcata aacggcctac gcttctcgaa attattcaac gccttaacga agccgaagac
     4621 atgaattata gtgcggcatc actctggcag gttcagtctg gtgatgaaga atcagatctc
     4681 tccgatactg aagcgttgga gacagagacc acgagcgagt ttcttccgtc cgatgaagaa
     4741 ccggcgtccg tcggtaagac cggcgagacc tctacacagg aaccggacaa acccgatctt
     4801 atatctaaac ttcccgcttc ggttgacctt tctgatctca aggtgcttga aaagataacc
     4861 gatgatttta gccatgagag aatagtcggg aaagacggga cttttaaagg atgccataag
     4921 gctttcgttt ataaaggtga cattcctctg agggagatga ttgcagttaa acggctcatt
     4981 ggtgttgaga tacccttcga gaagttcaag agagaggctg aacaattcat cagtttggat
     5041 cataagaaca tagttaaggt tgcaagttat tgccacgacc agagccgggg acatagactc
     5101 gtccagttca aggggaagcc tctgccccaa ctttttaatg ggcctgagca actgttgtgt
     5161 tacgagtaca tgcacaatgg ttctcttaga gattacttga tgggccaagg ctcccgggtc
     5221 atagattggc agatgcggta caagctgata aaagggacct gcgcaggctt gcattatctt
     5281 cataagggtc gcgcaggatg tccgatcgtt caccttaatc tgagtcccag caacgtgctt
     5341 ttggatcata attacattcc gcggataact ggcttcgact tttccaagct tataggagaa
     5401 aaaaacacta aatctgtggt cttgaagctc aatggaccca tcgcttatct gccccccgac
     5461 ttcttccaca gcaaaggtac agatctcaag tacctggcga cagttgatat atactctttg
     5521 ggcctgatga tactcgagat tgcaacacag caggaaatca agggcataca tggtgttctg
     5581 ataaagtcaa tcgaggaaaa ctggcgggaa gagtcccaaa tcacacgcct ctatacctct
     5641 cttggggctg atgagctgag gcaggtgaag atgtgtattg atataggact tgactgtgtc
     5701 aagagtaacc cagagaagcg ccccactgca ggagcaatta tgttgtggct tgataaagaa
     5761 tctaagccag tccccgtgtc acgggccgga gcgggcgttt tgccacggcc cccagttccg
     5821 accaacatca accatgcggg gcgcatccag ggtatcccaa tcgagaaatc acaagtcctt
     5881 attccttcat tcgcttgtaa tgaatatagc tctgtcaaaa ttcaaaattt gcacttgctg
     5941 ctctttatgg gtagcgacta caaggaccac gacggtgact acaaggacca cgacatcgac
     6001 tacaaggacg acgacgacaa gatctagacc cagctttctt gtacaaagtg gttgataaca
     6061 gcgggttaat taacatcttt tacccatacg atgttcctga ctatgcgggc tatccctatg
     6121 acgtcccgga ctatgcagga tcctatccat atgacgttcc agattacgct gctcagtgca
     6181 gcgcttagag ctcgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat
     6241 tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc
     6301 atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag
     6361 tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata
     6421 aattatcgcg cgcggtgtca tctatgttac tagatcggga attagcacta gaggatcccc
     6481 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     6541 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     6601 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
     6661 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     6721 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     6781 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     6841 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     6901 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     6961 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     7021 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     7081 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     7141 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     7201 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     7261 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     7321 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     7381 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     7441 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     7501 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     7561 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     7621 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     7681 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     7741 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     7801 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     7861 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     7921 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     7981 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     8041 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     8101 ccgtagaaaa gatcaaagga tcttc
//
