LOCUS       sequence-471109-        7960 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 7960)
  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 7960)
  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
FEATURES             Location/Qualifiers
     source          1..7960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             514..579
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(586..610)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             639..728
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      762..1014
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(1058..1075)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1058..1074)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1067..1089)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1283..1302)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1402..1424
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1462..1480)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1548..1652
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1653..1721,1722..2513)
                     /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(1871..1890)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2684..3272
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3173..3192
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3426..3443
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3560..3581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3596..3601,3602..3619,3620..3626)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3634..3650
                     /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     3639..3661
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3658..3674
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3658..3674
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5693..5717
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 gcttatctgc cccccgactt cttccacagc aaaggtacag atctcaagta cctggcgaca
       61 gttgatatat actctttggg cctgatgata ctcgagattg caacacagca ggaaatcaag
      121 ggcatacatg gtgttctgat aaagtcaatc gaggaaaact ggcgggaaga gtcccaaatc
      181 acacgcctct atacctctct tggggctgat gagctgaggc aggtgaagat gtgtattgat
      241 ataggacttg actgtgtcaa gagtaaccca gagaagcgcc ccactgcagg agcaattatg
      301 ttgtggcttg ataaagaatc taagccagtc cccgtgtcac gggccggagc gggcgttttg
      361 ccacggcccc cagttccgac caacatcaac catgcggggc gcatccaggg tatcccaatc
      421 gagaaatcac aagtccttat tccttcattc gcttgtaatg aatatagctc tgtcaaaatt
      481 caaaatttgc acttgctgct ctttatgggt agcgactaca aggaccacga cggtgactac
      541 aaggaccacg acatcgacta caaggacgac gacgacaaga tctagaccca gctttcttgt
      601 acaaagtggt tgataacagc gggttaatta acatctttta cccatacgat gttcctgact
      661 atgcgggcta tccctatgac gtcccggact atgcaggatc ctatccatat gacgttccag
      721 attacgctgc tcagtgcagc gcttagagct cgaatttccc cgatcgttca aacatttggc
      781 aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc
      841 tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat
      901 gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat
      961 agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat
     1021 tagcactaga ggatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc
     1081 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     1141 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     1201 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     1261 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     1321 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     1381 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     1441 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1501 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1561 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1621 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1681 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1741 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1801 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1861 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1921 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1981 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2041 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2101 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2161 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     2221 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2281 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2341 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2401 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2461 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2521 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2581 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2641 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2701 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2761 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2821 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2881 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2941 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3001 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3061 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3121 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3181 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3241 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3301 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3361 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3421 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     3481 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     3541 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     3601 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     3661 gaaacagcta tgaccatgat tacgccaagc tttgcagcgt gacccggtcg tgcccctctc
     3721 tagagataat gagcattgca tgtctaagtt ataaaaaatt accacatatt ttttttgtca
     3781 cacttgtttg aagtgcagtt tatctatctt tatacatata tttaaacttt actctacgaa
     3841 taatataatc tatagtacta caataatatc agtgttttag agaatcatat aaatgaacag
     3901 ttagacatgg tctaaaggac aattgagtat tttgacaaca ggactctaca gttttatctt
     3961 tttagtgtgc atgtgttctc cttttttttt gcaaatagct tcacctatat aatacttcat
     4021 ccattttatt agtacatcca tttagggttt agggttaatg gtttttatag actaattttt
     4081 ttagtacatc tattttattc tattttagcc tctaaattaa gaaaactaaa actctatttt
     4141 agttttttta tttaataatt tagatataaa atagaataaa ataaagtgac taaaaattaa
     4201 acaaataccc tttaagaaat taaaaaaact aaggaaacat ttttcttgtt tcgagtagat
     4261 aatgccagcc tgttaaacgc cgtcgacgag tctaacggac accaaccagc gaaccagcag
     4321 cgtcgcgtcg ggccaagcga agcagacggc acggcatctc tgtcgctgcc tctggacccc
     4381 tctcgagagt tccgctccac cgttggactt gctccgctgt cggcatccag aaattgcgtg
     4441 gcggagcggc agacgtgagc cggcacggca ggcggcctcc tcctcctctc acggcaccgg
     4501 cagctacggg ggattccttt cccaccgctc cttcgctttc ccttcctcgc ccgccgtaat
     4561 aaatagacac cccctccaca ccctctttcc ccaacctcgt gttgttcgga gcgcacacac
     4621 acacaaccag atctccccca aatccacccg tcggcacctc cgcttcaagg tacgccgctc
     4681 gtcctccccc cccccccctc tctaccttct ctagatcggc gttccggtcc atggttaggg
     4741 cccggtagtt ctacttctgt tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc
     4801 tgctagcgtt cgtacacgga tgcgacctgt acgtcagaca cgttctgatt gctaacttgc
     4861 cagtgtttct ctttggggaa tcctgggatg gctctagccg ttccgcagac gggatcgatt
     4921 tcatgatttt ttttgtttcg ttgcataggg tttggtttgc ccttttcctt tatttcaata
     4981 tatgccgtgc acttgtttgt cgggtcatct tttcatgctt ttttttgtct tggttgtgat
     5041 gatgtggtct ggttgggcgg tcgttctaga tcggagtaga attctgtttc aaactacctg
     5101 gtggatttat taattttgga tctgtatgtg tgtgccatac atattcatag ttacgaattg
     5161 aagatgatgg atggaaatat cgatctagga taggtataca tgttgatgcg ggttttactg
     5221 atgcatatac agagatgctt tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg
     5281 tcgttcattc gttctagatc ggagtagaat actgtttcaa actacctggt gtatttatta
     5341 attttggaac tgtatgtgtg tgtcatacat cttcatagtt acgagtttaa gatggatgga
     5401 aatatcgatc taggataggt atacatgttg atgtgggttt tactgatgca tatacatgat
     5461 ggcatatgca gcatctattc atatgctcta accttgagta cctatctatt ataataaaca
     5521 agtatgtttt ataattattt tgatcttgat atacttggat gatggcatat gcagcagcta
     5581 tatgtggatt tttttagccc tgccttcata cgctatttat ttgcttggta ctgtttcttt
     5641 tgtcgatgct caccctgttg tttggtgtta cttctgcaga ctagagttat caacaagttt
     5701 gtacaaaaaa gcaggctcca ccatgatggg tggctatgag ttccagaggg cagaattgga
     5761 cgcccttgag ggtgtagttc gtgacccaac cgccgaaccc atgtctttaa cactacctct
     5821 tttgcgtcac attacgaatg atttctcccc tgagttcgag atatctaagg acgactccgc
     5881 cgtggtatat ctaggagtgc taccatcagg atttagggtg gcagtgaaga aatcacattt
     5941 taggttttgt ctggacgacg aggacgcctt cactaatgaa gtttcaattg caatgaaagc
     6001 cgcacataag aatactgtaa gagtaatcgg ctattgtcat catacacatg agcagattgc
     6061 cgagtatgag ggaaagcaag tcttcgcaga agtgcgtgag aggttgatat gtactgagta
     6121 cgtgcccaat ggacctctat ccggccatat cgaaggcaaa atttgtgcac aaatggacgg
     6181 ctacgagttc cagcgtgctg agcttgatgc attggagaga gttgttagag atacctctgc
     6241 cgagccaatg tccttgacac tgcccctttt gcgtcatatc acaaatgatt tctcagatga
     6301 atccaggatc ggacgtggag gattcgctgt tgtctatctt ggtgttttgc catccggcct
     6361 gagaatagct gttaagaggt tgtcaaatat agcttatatg aatgaaagtg cattccagaa
     6421 cgaagtattc atcacaatga aagccacaca taaaaataca gtgagattca tgggatactg
     6481 ctcacagatt caaggcaaac taatagaaca tgatggacaa catgtctttg cacagcttga
     6541 agagaggcta atttgtgtgg aatacgcacc taagggaaca ttagacgctc acatcggaga
     6601 ttatggtgaa ctggactgga accaaagata tcagattctg aaaggcatat gtcagggttt
     6661 acatcatcta catgacgaaa tgcatgtctt tcatggagat atcaaacccg ccaacattct
     6721 tatcggcgat aacctagttc ctaaaatcta tgatttcgga ttgagtcaaa tgttcgagga
     6781 ggaggaaacg gaaagaattg tggaaaacat tgccggtaca ttcggctaca tggcacctga
     6841 gttctgtaca aacaacatgg tatccttcaa ggccgagatt tacagtttag gcgttgtaat
     6901 aggagaactt cttatcggta aaaaaggatg gtttgacgag gacgtgcgga agctttttgt
     6961 tcagcagttg aaaggccttc gcaaaacttt ggtgaaggaa ggcgcgtttt ccagctggga
     7021 gaataaatac catcaagtgc gcacgtgtat ggagattggt caggattgca tcgatccaaa
     7081 tcctcataaa cggcctacgc ttctcgaaat tattcaacgc cttaacgaag ccgaagacat
     7141 gaattatagt gcggcatcac tctggcaggt tcagtctggt gatgaagaat cagatctctc
     7201 cgatactgaa gcgttggaga cagagaccac gagcgagttt cttccgtccg atgaagaacc
     7261 ggcgtccgtc ggtaagaccg gcgagacctc tacacaggaa ccggacaaac ccgatcttat
     7321 atctaaactt cccgcttcgg ttgacctttc tgatctcaag gtgcttgaaa agataaccga
     7381 tgattttagc catgagagaa tagtcgggaa agacgggact tttaaaggat gccataaggc
     7441 tttcgtttat aaaggtgaca ttcctctgag ggagatgatt gcagttaaac ggctcattgg
     7501 tgttgagata cccttcgaga agttcaagag agaggctgaa caattcatca gtttggatca
     7561 taagaacata gttaaggttg caagttattg ccacgaccag agccggggac atagactcgt
     7621 ccagttcaag gggaagcctc tgccccaact ttttaatggg cctgagcaac tgttgtgtta
     7681 cgagtacatg cacaatggtt ctcttagaga ttacttgatg ggccaaggct cccgggtcat
     7741 agattggcag atgcggtaca agctgataaa agggacctgc gcaggcttgc attatcttca
     7801 taagggtcgc gcaggatgtc cgatcgttca ccttaatctg agtcccagca acgtgctttt
     7861 ggatcataat tacattccgc ggataactgg cttcgacttt tccaagctta taggagaaaa
     7921 aaacactaaa tctgtggtct tgaagctcaa tggacccatc
//
