LOCUS       sequence-470242-        7957 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protoplast expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7957)
  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 7957)
  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: pUC19::ZmUBI-RWT4Ca+C-Tail
FEATURES             Location/Qualifiers
     source          1..7957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1703..1727
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4523..4546
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(4553..4577)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4606..4695
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      4729..4981
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(5025..5042)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5025..5041)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5034..5056)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5250..5269)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5369..5391
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5429..5447)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5515..5619
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5620..5688,5689..6480)
                     /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(5838..5857)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6651..7239
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7140..7159
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7393..7410
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7527..7548
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7563..7568,7569..7586,7587..7593)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7601..7617
                     /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     7606..7628
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7625..7641
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7625..7641
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gcaaatagct tcacctatat aatacttcat ccattttatt agtacatcca tttagggttt
       61 agggttaatg gtttttatag actaattttt ttagtacatc tattttattc tattttagcc
      121 tctaaattaa gaaaactaaa actctatttt agttttttta tttaataatt tagatataaa
      181 atagaataaa ataaagtgac taaaaattaa acaaataccc tttaagaaat taaaaaaact
      241 aaggaaacat ttttcttgtt tcgagtagat aatgccagcc tgttaaacgc cgtcgacgag
      301 tctaacggac accaaccagc gaaccagcag cgtcgcgtcg ggccaagcga agcagacggc
      361 acggcatctc tgtcgctgcc tctggacccc tctcgagagt tccgctccac cgttggactt
      421 gctccgctgt cggcatccag aaattgcgtg gcggagcggc agacgtgagc cggcacggca
      481 ggcggcctcc tcctcctctc acggcaccgg cagctacggg ggattccttt cccaccgctc
      541 cttcgctttc ccttcctcgc ccgccgtaat aaatagacac cccctccaca ccctctttcc
      601 ccaacctcgt gttgttcgga gcgcacacac acacaaccag atctccccca aatccacccg
      661 tcggcacctc cgcttcaagg tacgccgctc gtcctccccc cccccccctc tctaccttct
      721 ctagatcggc gttccggtcc atggttaggg cccggtagtt ctacttctgt tcatgtttgt
      781 gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt cgtacacgga tgcgacctgt
      841 acgtcagaca cgttctgatt gctaacttgc cagtgtttct ctttggggaa tcctgggatg
      901 gctctagccg ttccgcagac gggatcgatt tcatgatttt ttttgtttcg ttgcataggg
      961 tttggtttgc ccttttcctt tatttcaata tatgccgtgc acttgtttgt cgggtcatct
     1021 tttcatgctt ttttttgtct tggttgtgat gatgtggtct ggttgggcgg tcgttctaga
     1081 tcggagtaga attctgtttc aaactacctg gtggatttat taattttgga tctgtatgtg
     1141 tgtgccatac atattcatag ttacgaattg aagatgatgg atggaaatat cgatctagga
     1201 taggtataca tgttgatgcg ggttttactg atgcatatac agagatgctt tttgttcgct
     1261 tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc gttctagatc ggagtagaat
     1321 actgtttcaa actacctggt gtatttatta attttggaac tgtatgtgtg tgtcatacat
     1381 cttcatagtt acgagtttaa gatggatgga aatatcgatc taggataggt atacatgttg
     1441 atgtgggttt tactgatgca tatacatgat ggcatatgca gcatctattc atatgctcta
     1501 accttgagta cctatctatt ataataaaca agtatgtttt ataattattt tgatcttgat
     1561 atacttggat gatggcatat gcagcagcta tatgtggatt tttttagccc tgccttcata
     1621 cgctatttat ttgcttggta ctgtttcttt tgtcgatgct caccctgttg tttggtgtta
     1681 cttctgcaga ctagagttat caacaagttt gtacaaaaaa gcaggctcca ccatgatggg
     1741 tggctatgag ttccagaggg cagaattgga cgcccttgag ggtgtagttc gtgacccaac
     1801 cgccgaaccc atgtctttaa cactacctct tttgcgtcac attacgaatg atttctcccc
     1861 tgagttcgag atatctaagg acgactccgc cgtggtatat ctaggagtgc taccatcagg
     1921 atttagggtg gcagtgaaga aatcacattt taggttttgt ctggacgacg aggacgcctt
     1981 cactaatgaa gtttcaattg caatgaaagc cgcacataag aatactgtaa gagtaatcgg
     2041 ctattgtcat catacacatg agcagattgc cgagtatgag ggaaagcaag tcttcgcaga
     2101 agtgcgtgag aggttgatat gtactgagta cgtgcccaat ggacctctat ccggccatat
     2161 cgaaggcaaa atttgtgcac aaatggacgg ctatgagttc cagcgtgctg agcttgatgc
     2221 attggagaga gttgttagag atacctctgc cgagccaatg tccttgacac tgcccctttt
     2281 gcgtcatatc acaaatgatt tctcagatga atccaggatc ggacgtggag gattcgctgt
     2341 tgtctatctt ggtgttttgc catccggcct gagaatagct gttaagaggt tgtcaaatat
     2401 agcttatatg aatgaaagtg cattccagaa cgaagtattc atcacaatga aagccacaca
     2461 taaaaataca gtgagattca tgggatactg ctcacagatt caaggcaaac taatagaaca
     2521 tgatggacaa catgtctttg cacagcttga agagaggcta atttgtgtgg aatacgcacc
     2581 taagggaaca ttagacgctc acatcggaga ttatggtgaa ctggactgga accaaagata
     2641 tcagattctg aaaggcatat gtcagggttt acatcatcta catgacgaaa tgcatgtctt
     2701 tcatggagat atcaaacccg ccaacattct tatcggcgat aacctagttc ctaaaatcta
     2761 tgatttcgga ttgagtcaaa tgttcgagga ggaggaaacg gaaagaattg tggaaaacat
     2821 tgccggtaca ttcggctaca tggcacctga gttctgtaca aacaacatgg tatccttcaa
     2881 ggccgagatt tacagtttag gcgttgtaat aggagaactt cttatcggta aaaaaggatg
     2941 gtttgacgag gacgtgcgga agctttttgt tcagcagttg aaaggccttc gcaaaacttt
     3001 ggtgaaggaa ggcgcgtttt ccagctggga gaataaatac catcaagtgc gcacgtgtat
     3061 ggagattggt caggattgca tcgatccaaa tcctcataaa cggcctacgc ttctcgaaat
     3121 tattcaacgc cttaacgaag ccgaagacat gaattatagt gcggcatcac tctggcaggt
     3181 tcagtctggt gatgaagaat cagatctctc cgatactgaa gcgttggaga cagagaccac
     3241 gagcgagttt cttccgtccg atgaagaacc ggcgtccgtc ggtaagaccg gcgagacctc
     3301 tacacaggaa ccggacaaac ccgatcttat atctaaactt cccgcttcgg ttgacctttc
     3361 tgatctcaag gtgcttgaaa agataaccga tgattttagc catgagagaa tagtcgggaa
     3421 agacgggact tttaaaggat gccataaggc tttcgtttat aaaggtgaca ttcctctgag
     3481 ggagatgatt gcagttaaac ggctcattgg tgttgagata cccttcgaga agttcaagag
     3541 agaggctgaa caattcatca gtttggatca taagaacata gttaaggttg caagttattg
     3601 ccacgaccag agccggggac atagactcgt ccagttcaag gggaagcctc tgccccaact
     3661 ttttaatggg cctgagcaac tgttgtgtta cgagtacatg cacaatggtt ctcttagaga
     3721 ttacttgatg ggccaaggct cccgggtcat agattggcag atgcggtaca agctgataaa
     3781 agggacctgc gcaggcttgc attatcttca taagggtcgc gcaggatgtc cgatcgttca
     3841 ccttaatctg agtcccagca acgtgctttt ggatcataat tacattccgc ggataactgg
     3901 cttcgacttt tccaagctta taggagaaaa aaacactaaa tctgtggtct tgaagctcaa
     3961 tggacccatc gcttatctgc cccccgactt cttccacagc aaaggtacag atctcaagta
     4021 cctggcgaca gttgatatat actctttggg cctgatgata ctcgagattg caacacagca
     4081 ggaaatcaag ggcatacatg gtgttctgat aaagtcaatc gaggaaaact ggcgggaaga
     4141 gtcccaaatc acacgcctct atacctctct tggggctgat gagctgaggc aggtgaagat
     4201 gtgtattgat ataggacttg actgtgtcaa gagtaaccca gagaagcgcc ccactgcagg
     4261 agcaattatg ttgtggcttg ataaagaatc taagccagtc cccgtgtcac gggccggagc
     4321 gggcgttttg ccacggcccc cagttccgac caacatcaac catgcggggc gcatccaggg
     4381 tatcccaatc gagaaatcac aagtccttat tccttcattc gcttgtaatg aatatagctc
     4441 tgtcaaaatt caaaatttgc acttgctgct ctttatgttc atacaagttg accaaggtag
     4501 cgactacaag gaccacgacg gtgactacaa ggacgacgac gacaagatct agacccagct
     4561 ttcttgtaca aagtggttga taacagcggg ttaattaaca tcttttaccc atacgatgtt
     4621 cctgactatg cgggctatcc ctatgacgtc ccggactatg caggatccta tccatatgac
     4681 gttccagatt acgctgctca gtgcagcgct tagagctcga atttccccga tcgttcaaac
     4741 atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata
     4801 taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt
     4861 atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac
     4921 aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat
     4981 cgggaattag cactagagga tccccgggta ccgagctcga attcactggc cgtcgtttta
     5041 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     5101 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     5161 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     5221 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5281 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5341 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5401 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5461 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5521 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5581 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5641 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5701 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5761 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5821 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5881 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5941 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6001 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6061 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6121 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6181 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6241 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6301 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6361 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6421 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6481 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6541 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6601 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6661 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6721 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6781 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6841 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6901 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6961 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7021 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7081 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7141 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7201 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7261 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     7321 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     7381 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     7441 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     7501 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     7561 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     7621 cacacaggaa acagctatga ccatgattac gccaagcttt gcagcgtgac ccggtcgtgc
     7681 ccctctctag agataatgag cattgcatgt ctaagttata aaaaattacc acatattttt
     7741 tttgtcacac ttgtttgaag tgcagtttat ctatctttat acatatattt aaactttact
     7801 ctacgaataa tataatctat agtactacaa taatatcagt gttttagaga atcatataaa
     7861 tgaacagtta gacatggtct aaaggacaat tgagtatttt gacaacagga ctctacagtt
     7921 ttatcttttt agtgtgcatg tgttctcctt ttttttt
//
