LOCUS       sequence-472255-        7531 bp    DNA     circular SYN 09-MAY-2025
DEFINITION  Protoplast expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7531)
  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 7531)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 9, 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_noKDup
FEATURES             Location/Qualifiers
     source          1..7531
                     /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             5365..5430
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(5434..5458)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5487..5576
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      5610..5862
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(5906..5923)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5906..5922)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5915..5937)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6131..6150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6250..6272
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6310..6328)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6396..6500
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6501..6569,6570..7361)
                     /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(6719..6738)
                     /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 tggaaggcaa aatttgtgca
     3061 caaatggacg gctacgagtt ccagcgtgct gagcttgatg cattggagag agttgttaga
     3121 gatacctctg ccgagccaat gtccttgaca ctgccccttt tgcgtcatat cacaaatgat
     3181 ttctcagatg aatccaggat cggacgtgga ggattcgctg ttgtctatct tggtgttttg
     3241 ccatccggcc tgagaatagc tgttaagagg ttgtcaaata tagcttatat gaatgaaagt
     3301 gcattccaga acgaagtatt catcacaatg aaagccacac ataaaaatac agtgagattc
     3361 atgggatact gctcacagat tcaaggcaaa ctaatagaac atgatggaca acatgtcttt
     3421 gcacagcttg aagagaggct aatttgtgtg gaatacgcac ctaagggaac attagacgct
     3481 cacatcggag attatggtga actggactgg aaccaaagat atcagattct gaaaggcata
     3541 tgtcagggtt tacatcatct acatgacgaa atgcatgtct ttcatggaga tatcaaaccc
     3601 gccaacattc ttatcggcga taacctagtt cctaaaatct atgatttcgg attgagtcaa
     3661 atgttcgagg aggaggaaac ggaaagaatt gtggaaaaca ttgccggtac attcggctac
     3721 atggcacctg agttctgtac aaacaacatg gtatccttca aggccgagat ttacagttta
     3781 ggcgttgtaa taggagaact tcttatcggt aaaaaaggat ggtttgacga ggacgtgcgg
     3841 aagctttttg ttcagcagtt gaaaggcctt cgcaaaactt tggtgaagga aggcgcgttt
     3901 tccagctggg agaataaata ccatcaagtg cgcacgtgta tggagattgg tcaggattgc
     3961 atcgatccaa atcctcataa acggcctacg cttctcgaaa ttattcaacg ccttaacgaa
     4021 gccgaagaca tgaattatag tgcggcatca ctctggcagg ttcagtctgg tgatgaagaa
     4081 tcagatctct ccgatactga agcgttggag acagagacca cgagcgagtt tcttccgtcc
     4141 gatgaagaac cggcgtccgt cggtaagacc ggcgagacct ctacacagga accggacaaa
     4201 cccgatctta tatctaaact tcccgcttcg gttgaccttt ctgatctcaa ggtgcttgaa
     4261 aagataaccg atgattttag ccatgagaga atagtcggga aagacgggac ttttaaagga
     4321 tgccataagg ctttcgttta taaaggtgac attcctctga gggagatgat tgcagttaaa
     4381 cggctcattg gtgttgagat acccttcgag aagttcaaga gagaggctga acaattcatc
     4441 agtttggatc ataagaacat agttaaggtt gcaagttatt gccacgacca gagccgggga
     4501 catagactcg tccagttcaa ggggaagcct ctgccccaac tttttaatgg gcctgagcaa
     4561 ctgttgtgtt acgagtacat gcacaatggt tctcttagag attacttgat gggccaaggc
     4621 tcccgggtca tagattggca gatgcggtac aagctgataa aagggacctg cgcaggcttg
     4681 cattatcttc ataagggtcg cgcaggatgt ccgatcgttc accttaatct gagtcccagc
     4741 aacgtgcttt tggatcataa ttacattccg cggataactg gcttcgactt ttccaagctt
     4801 ataggagaaa aaaacactaa atctgtggtc ttgaagctca atggacccat cgcttatctg
     4861 ccccccgact tcttccacag caaaggtaca gatctcaagt acctggcgac agttgatata
     4921 tactctttgg gcctgatgat actcgagatt gcaacacagc aggaaatcaa gggcatacat
     4981 ggtgttctga taaagtcaat cgaggaaaac tggcgggaag agtcccaaat cacacgcctc
     5041 tatacctctc ttggggctga tgagctgagg caggtgaaga tgtgtattga tataggactt
     5101 gactgtgtca agagtaaccc agagaagcgc cccactgcag gagcaattat gttgtggctt
     5161 gataaagaat ctaagccagt ccccgtgtca cgggccggag cgggcgtttt gccacggccc
     5221 ccagttccga ccaacatcaa ccatgcgggg cgcatccagg gtatcccaat cgagaaatca
     5281 caagtcctta ttccttcatt cgcttgtaat gaatatagct ctgtcaaaat tcaaaatttg
     5341 cacttgctgc tctttatggg tagcgactac aaggaccacg acggtgacta caaggaccac
     5401 gacatcgact acaaggacga cgacgacaag atcacccagc tttcttgtac aaagtggttg
     5461 ataacagcgg gttaattaac atcttttacc catacgatgt tcctgactat gcgggctatc
     5521 cctatgacgt cccggactat gcaggatcct atccatatga cgttccagat tacgctgctc
     5581 agtgcagcgc ttagagctcg aatttccccg atcgttcaaa catttggcaa taaagtttct
     5641 taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg
     5701 ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga
     5761 ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact
     5821 aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggaatta gcactagagg
     5881 atccccgggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     5941 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     6001 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     6061 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     6121 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     6181 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     6241 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     6301 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     6361 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     6421 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     6481 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     6541 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6601 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6661 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6721 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6781 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6841 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6901 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6961 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     7021 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     7081 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     7141 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     7201 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     7261 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     7321 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     7381 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     7441 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7501 cagaccccgt agaaaagatc aaaggatctt c
//
