LOCUS       sequence-471114-        5422 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 5422)
  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 5422)
  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-VirPWT4
FEATURES             Location/Qualifiers
     source          1..5422
                     /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             3253..3318
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(3325..3349)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3378..3467
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      3501..3753
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(3797..3814)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3797..3813)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3806..3828)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4022..4041)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4141..4163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4201..4219)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4287..4391
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4392..4460,4461..5252)
                     /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(4610..4629)
                     /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 tgctgaattc cccagaagtc
     3061 ccgacttata aaattggaca tcctgggcct cggtatatta gatataacga tggtgaaatc
     3121 catgaggtcg aacgcaagga atcagggcgc tatcgagtag acgacggggg tttaatacgt
     3181 gaggaatatg aaaaaaaaac tcaagattcg gaagggtcaa aaactcccac cacgcgcggt
     3241 ccgaaaggta gcgactacaa ggaccacgac ggtgactaca aggaccacga catcgactac
     3301 aaggacgacg acgacaagat ctagacccag ctttcttgta caaagtggtt gataacagcg
     3361 ggttaattaa catcttttac ccatacgatg ttcctgacta tgcgggctat ccctatgacg
     3421 tcccggacta tgcaggatcc tatccatatg acgttccaga ttacgctgct cagtgcagcg
     3481 cttagagctc gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga
     3541 atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg
     3601 taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc
     3661 cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat
     3721 tatcgcgcgc ggtgtcatct atgttactag atcgggaatt agcactagag gatccccggg
     3781 taccgagctc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     3841 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     3901 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga
     3961 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
     4021 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     4081 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4141 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     4201 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     4261 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4321 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4381 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4441 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4501 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4561 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4621 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4681 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4741 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4801 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4861 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4921 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4981 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5041 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5101 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5161 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5221 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5281 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     5341 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5401 tagaaaagat caaaggatct tc
//
