LOCUS       sequence-471101-        7942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Recombinant protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7942)
  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 7942)
  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: pET28a:His-MBP-KD
FEATURES             Location/Qualifiers
     source          1..7942
                     /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"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3485..3504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3504..3528
                     /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)."
     RBS             3543..3565
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3584..3601
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3611..4711
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     4685..4708
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4766..4786
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             6290..6307
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6360..6378)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6374..6421
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6458..6913
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6545..6564)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6755..6776
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7006..7821)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(7006..7821)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     7729..7748
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7813..7832
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct 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 agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat accctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     3541 attttgttta actttaagaa ggagatatac catgggttct tctcaccatc accatcacca
     3601 tggttcttct atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg
     3661 ctataacggt ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac
     3721 cgttgagcat ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg
     3781 ccctgacatt atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt
     3841 ggctgaaatc accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc
     3901 cgtacgttac aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat
     3961 ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga
     4021 taaagaactg aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt
     4081 cacctggccg ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta
     4141 cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt
     4201 tgacctgatt aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc
     4261 ctttaataaa ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga
     4321 caccagcaaa gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa
     4381 accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc
     4441 aaaagagttc ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga
     4501 caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg
     4561 tattgccgcc actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat
     4621 gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac
     4681 tgtcgatgaa gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa
     4741 caataacaac aacctcggga tcgaggaaaa cctgtacttc caatccaatg ccatgggggg
     4801 atatgaattc cagagagcag aattggacgc ccttgagggt gtagttcgtg acccaaccgc
     4861 cgaacccatg tctttaacac tacctctttt gcgtcacatt acgaatgatt tctcccctga
     4921 gttcgagata tctaaggacg actccgccgt ggtatatcta ggagtgctac catcaggatt
     4981 tagggtggca gtgaagaaat cacattttag gttttgtctg gacgacgagg acgccttcac
     5041 taatgaagtt tcaattgcaa tgaaagccgc acataagaat actgtaagag taatcggcta
     5101 ttgtcatcat acacatgagc agattgccga gtatgaggga aagcaagtct tcgcagaagt
     5161 gcgtgagagg ttgatatgta ctgagtacgt gcccaatgga cctctatccg gccatatcga
     5221 aggcaaaatt tgtgcacaaa tggacggcta cgagttccag cgtgctgagc ttgatgcatt
     5281 ggagagagtt gttagagata cctctgccga gccaatgtcc ttgacactgc cccttttgcg
     5341 tcatatcaca aatgatttct cagatgaatc caggatcgga cgtggaggat tcgctgttgt
     5401 ctatcttggt gttttgccat ccggcctgag aatagctgtt aagaggttgt caaatatagc
     5461 ttatatgaat gaaagtgcat tccagaacga agtattcatc acaatgaaag ccacacataa
     5521 aaatacagtg agattcatgg gatactgctc acagattcaa ggcaaactaa tagaacatga
     5581 tggacaacat gtctttgcac agcttgaaga gaggctaatt tgtgtggaat acgcacctaa
     5641 gggaacatta gacgctcaca tcggagatta tggtgaactg gactggaacc aaagatatca
     5701 gattctgaaa ggcatatgtc agggtttaca tcatctacat gacgaaatgc atgtctttca
     5761 tggagatatc aaacccgcca acattcttat cggcgataac ctagttccta aaatctatga
     5821 tttcggattg agtcaaatgt tcgaggagga ggaaacggaa agaattgtgg aaaacattgc
     5881 cggtacattc ggctacatgg cacctgagtt ctgtacaaac aacatggtat ccttcaaggc
     5941 cgagatttac agtttaggcg ttgtaatagg agaacttctt atcggtaaaa aaggatggtt
     6001 tgacgaggac gtgcggaagc tttttgttca gcagttgaaa ggccttcgca aaactttggt
     6061 gaaggaaggc gcgttttcca gctgggagaa taaataccat caagtgcgca cgtgtatgga
     6121 gattggtcag gattgcatcg atccaaatcc tcataaacgg cctacgcttc tcgaaattat
     6181 tcaacgcctt aacgaagccg aagacatgaa ttatagtgcg gcataacatt ggaagtggat
     6241 aacggatccg aattcgagcg ccgtcgacaa gcttgcggcc gcactcgagc accaccacca
     6301 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     6361 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     6421 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
     6481 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     6541 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     6601 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     6661 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     6721 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     6781 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     6841 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaactagta
     6901 acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6961 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     7021 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     7081 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     7141 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     7201 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     7261 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     7321 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     7381 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     7441 gaacactgcc agcgcatcaa caatgttttc acctgaatca ggatattctt ctaatacctg
     7501 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     7561 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     7621 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     7681 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     7741 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     7801 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     7861 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     7921 tagaaaagat caaaggatct tc
//
