LOCUS       sequence-472267-        5442 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 5442)
  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 5442)
  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-AvrPWT4
FEATURES             Location/Qualifiers
     source          1..5442
                     /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    3009..3033
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3273..3338
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(3345..3369)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3398..3487
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      3521..3773
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(3817..3834)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3817..3833)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3826..3848)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4042..4061)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4161..4183
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4221..4239)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4307..4411
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4412..4480,4481..5272)
                     /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(4630..4649)
                     /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 cccctctcta ccttctctag atcggcgttc
     2041 cggtccatgg ttagggcccg gtagttctac ttctgttcat gtttgtgtta gatccgtgtt
     2101 tgtgttagat ccgtgctgct agcgttcgta cacggatgcg acctgtacgt cagacacgtt
     2161 ctgattgcta acttgccagt gtttctcttt ggggaatcct gggatggctc tagccgttcc
     2221 gcagacggga tcgatttcat gatttttttt gtttcgttgc atagggtttg gtttgccctt
     2281 ttcctttatt tcaatatatg ccgtgcactt gtttgtcggg tcatcttttc atgctttttt
     2341 ttgtcttggt tgtgatgatg tggtctggtt gggcggtcgt tctagatcgg agtagaattc
     2401 tgtttcaaac tacctggtgg atttattaat tttggatctg tatgtgtgtg ccatacatat
     2461 tcatagttac gaattgaaga tgatggatgg aaatatcgat ctaggatagg tatacatgtt
     2521 gatgcgggtt ttactgatgc atatacagag atgctttttg ttcgcttggt tgtgatgatg
     2581 tggtgtggtt gggcggtcgt tcattcgttc tagatcggag tagaatactg tttcaaacta
     2641 cctggtgtat ttattaattt tggaactgta tgtgtgtgtc atacatcttc atagttacga
     2701 gtttaagatg gatggaaata tcgatctagg ataggtatac atgttgatgt gggttttact
     2761 gatgcatata catgatggca tatgcagcat ctattcatat gctctaacct tgagtaccta
     2821 tctattataa taaacaagta tgttttataa ttattttgat cttgatatac ttggatgatg
     2881 gcatatgcag cagctatatg tggatttttt tagccctgcc ttcatacgct atttatttgc
     2941 ttggtactgt ttcttttgtc gatgctcacc ctgttgtttg gtgttacttc tgcagactag
     3001 agttatcaac aagtttgtac aaaaaagcag gctccaccat gaaacccccc tccgattcga
     3061 cggatgcctc aaaaatcccg acttacaaaa ttaaacatcc agggcctcgt tacattataa
     3121 ctgacgatgg tcgagttgtt gaggtcgaac gcaaggaatc aggacgctat caattaatcg
     3181 atggggtttt agtacgtgtg gaatatgaaa agaaaaccgg gggttcggaa gggtcaaaaa
     3241 catccaccaa cacgcgcggt ccgaaaggta gcgactacaa ggaccacgac ggtgactaca
     3301 aggaccacga catcgactac aaggacgacg acgacaagat ctagacccag ctttcttgta
     3361 caaagtggtt gataacagcg ggttaattaa catcttttac ccatacgatg ttcctgacta
     3421 tgcgggctat ccctatgacg tcccggacta tgcaggatcc tatccatatg acgttccaga
     3481 ttacgctgct cagtgcagcg cttagagctc gaatttcccc gatcgttcaa acatttggca
     3541 ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct
     3601 gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg
     3661 ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata
     3721 gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt
     3781 agcactagag gatccccggg taccgagctc gaattcactg gccgtcgttt tacaacgtcg
     3841 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     3901 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     3961 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4021 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     4081 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4141 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4201 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4261 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4321 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4381 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4441 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4501 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4561 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4621 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4681 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4741 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4801 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4861 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4921 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4981 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5041 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5101 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5161 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5221 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5281 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5341 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5401 ccactgagcg tcagaccccg tagaaaagat caaaggatct tc
//
