LOCUS       sequence-472258-        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_Reg2
FEATURES             Location/Qualifiers
     source          1..7960
                     /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             4484..4549
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(4556..4580)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4609..4698
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      4732..4984
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(5028..5045)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5028..5044)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5037..5059)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5253..5272)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5372..5394
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5432..5450)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5518..5622
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5623..5691,5692..6483)
                     /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(5841..5860)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6654..7242
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7143..7162
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7396..7413
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7530..7551
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7566..7571,7572..7589,7590..7596)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7604..7620
                     /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     7609..7631
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7628..7644
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7628..7644
                     /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 ctacgagttc 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 agcactggac 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 ctttatgggt agcgactaca aggaccacga
     4501 cggtgactac aaggaccacg acatcgacta caaggacgac gacgacaaga tctagaccca
     4561 gctttcttgt acaaagtggt tgataacagc gggttaatta acatctttta cccatacgat
     4621 gttcctgact atgcgggcta tccctatgac gtcccggact atgcaggatc ctatccatat
     4681 gacgttccag attacgctgc tcagtgcagc gcttagagct cgaatttccc cgatcgttca
     4741 aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc
     4801 atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta
     4861 tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa
     4921 aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta
     4981 gatcgggaat tagcactaga ggatccccgg gtaccgagct cgaattcact ggccgtcgtt
     5041 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     5101 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     5161 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5221 ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5281 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5341 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5401 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5461 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5521 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5581 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5641 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5701 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5761 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5821 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5881 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5941 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6001 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6061 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6121 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6181 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6241 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6301 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6361 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6421 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6481 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6541 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6601 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6661 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6721 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6781 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6841 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6901 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6961 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7021 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7081 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7141 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7201 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7261 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     7321 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     7381 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     7441 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     7501 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     7561 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     7621 tttcacacag gaaacagcta tgaccatgat tacgccaagc tttgcagcgt gacccggtcg
     7681 tgcccctctc tagagataat gagcattgca tgtctaagtt ataaaaaatt accacatatt
     7741 ttttttgtca cacttgtttg aagtgcagtt tatctatctt tatacatata tttaaacttt
     7801 actctacgaa taatataatc tatagtacta caataatatc agtgttttag agaatcatat
     7861 aaatgaacag ttagacatgg tctaaaggac aattgagtat tttgacaaca ggactctaca
     7921 gttttatctt tttagtgtgc atgtgttctc cttttttttt
//
