LOCUS       sequence-471106-        7945 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 7945)
  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 7945)
  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-RWT4Cs
FEATURES             Location/Qualifiers
     source          1..7945
                     /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             5818..5841
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(5848..5872)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5901..5990
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      6024..6276
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(6320..6337)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6320..6336)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6329..6351)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6545..6564)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6664..6686
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6724..6742)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6810..6914
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6915..6983,6984..7775)
                     /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(7133..7152)
                     /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 tgatgggtgg ctatgaattt
     3061 caacgagcag aattggacgc tctggaggga gtcgtcagag acccaactgc agaaccaatg
     3121 tcccttacat tacccctttt acgtcacata acgaatgact tttccccaga gtttgagatc
     3181 agcaaggatg attccgccgt ggtttatctc ggcgttctcc caagtggttt cagagtcgca
     3241 gtgaagaagc tccataaccg acattggctg gaagatgagg atgcctttat taatgaggta
     3301 agtatcgcta tgaaggccgc taaaaagaac acggtccgtg ttattggcta ttgtcagcat
     3361 acgcaagagc aaatcgcaga atacgaagga aaacaagtgt tcgccgagct tacggagcgt
     3421 ttgatctgca ccgaatacgt ccccaacgga ccgctctctg gccacattga ggggaaaata
     3481 tgtgcacaaa tggacggata tgagtttcaa agggcagagt tggacgcact ggaaagagtc
     3541 gtacgagaca cttcagctga accgatgtcc ttgactcttc cgttgctcag acatattacg
     3601 aatgactttt ccgacgaaag ccgaataggc aggggaggct ttgccgtggt atacctggga
     3661 gttttaccta gcggtttgag gattgctgtt aagaggctta gtaatattgc ctacatgaat
     3721 gaatcagctt ttcaaaacga ggttttcatt acgatgaaag ccacccataa aaataccgtc
     3781 cgattcatgg gctattgctc acaaatacag ggaaaattaa tagagcacaa tgggcagcat
     3841 gtctttgctc agttagagga gaggctgatt tgtgtcgaat acgctccaaa aggcacttta
     3901 gacgcacata taggggacta cggggagctc gactggaacc aacgatacca gatacttaag
     3961 gggatatgtc aaggcctcca ccacctccac gatgagatgc atgtctttca cggtgatatc
     4021 aagcctgcca atatcttgat tggtgacaat ctcgtcccca aaatctacga ttttggactt
     4081 tcacaaatgt ttgaagaaga agagacggag cgaatcgtaa aaaatatggc tggtacgttg
     4141 tgctacctcg caccggaggt tcttaacacc catatgatgt cttttaaggc tgagatatat
     4201 tctttaggtg tggtgattga ggaattactg acgggtaaga agggctggat cgatgaagac
     4261 gttagaaagc aactcaaggg cctcagaaag acattagtga aggaaggtgc tttttcaagt
     4321 tgggagaaca aataccatca ggtaaggacc tgcatggaaa ttggtcagga ctgtatcgat
     4381 cccaatccgc ataagaggcc caccctgttc gaaattattc agcgtcttaa tgaagctgag
     4441 gacatgaatt acagtgccgc aagcttgtgg caggttcagt ctggtgacga agaatcagac
     4501 ttgtctgaca cggaagcatt agaaaccgaa acaactagcg agttcctccc gtcagacgag
     4561 gagccggcaa gtgttggtaa gactggggag acttctactc aggaacccga taagccagac
     4621 ttggtttcaa agctgccagc aagcgtcgat ctgtccgatt taaaagtgct cgaaaaaata
     4681 acagatgact tcagccatga gagaatcgtt ggcaaggacg gtacattcaa gggttgccac
     4741 aaagcattcg tgtataaggg cgacattccg cgacgtgaga tgatcgccgt caaaagactc
     4801 atcggtgttg aaatacccgt cgaaaagttc aagagggagg ctgaacagtt cattcgttta
     4861 gatcataaga acattgttaa agtcgcatcc tattgccacg accaatccac tggtcacagg
     4921 ctcgtgcagt ttaaaggaaa accattgccc cagttattta acggtcctga acagttgctc
     4981 tgctatgagt atatgcataa tgggtctttg cgtgactatc ttatgggtca gggtagccgt
     5041 gaaatcgatt ggcagatgcg ttataaactg atcaaaggga cttgcgcagg ccttgattac
     5101 cttcataagg ggcgagctgg ttgcccgatt gtccatctca acttaagccc gagcaacgtt
     5161 ttattggatc ataactacat acctaggatt accggcttcg atttctctaa gttgattgga
     5221 gaaaaaaata cgaagagtgt agttctcaaa ctcaacggac caattgccta tcttcctccg
     5281 gattttttcc acagtaaagg caccgacttg aagtacctgg caaccgtcga catttactca
     5341 cttgggttga tgatcctgga aattgctacg caacaagaaa ttaagggaat tcacggagtg
     5401 ctgatcaaga gtatcgaaga gaactggagg gaggagagtc agataacccg attgtatacc
     5461 tccttgggag ctgatgaatt acgtcaagtg aaaatgtgca ttgatatcgg cctggactgc
     5521 gttaagtcaa accctgaaaa aaggcctacc gcaggggcta taatgctctg gttggataag
     5581 gaatctaaac ccgtaccagt atcaagggct ggggccggag tcttaccacg accccctgta
     5641 cctactaaca taaaccacgc tggtaggata cagggaatac caattgaaaa aagccaggtc
     5701 ttaatcccta gcttcgcatg taacgaatat tcctcagtta agattcaaaa cctgcacctt
     5761 ttgctcttca tgttcataca agttgaccaa ggtagcgact acaaggacca cgacggtgac
     5821 tacaaggacg acgacgacaa gatctagacc cagctttctt gtacaaagtg gttgataaca
     5881 gcgggttaat taacatcttt tacccatacg atgttcctga ctatgcgggc tatccctatg
     5941 acgtcccgga ctatgcagga tcctatccat atgacgttcc agattacgct gctcagtgca
     6001 gcgcttagag ctcgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat
     6061 tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc
     6121 atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag
     6181 tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata
     6241 aattatcgcg cgcggtgtca tctatgttac tagatcggga attagcacta gaggatcccc
     6301 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     6361 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     6421 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
     6481 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     6541 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     6601 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     6661 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     6721 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     6781 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     6841 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     6901 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     6961 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     7021 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     7081 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     7141 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     7201 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     7261 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     7321 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     7381 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     7441 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     7501 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     7561 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     7621 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     7681 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     7741 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     7801 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     7861 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     7921 ccgtagaaaa gatcaaagga tcttc
//
