LOCUS       sequence-471103-        8088 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 8088)
  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 8088)
  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-RWT4Ca
FEATURES             Location/Qualifiers
     source          1..8088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             617..634
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(687..705)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      701..748
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      785..1240
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(872..891)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1082..1103
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1333..2148)
                     /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(1333..2148)
                     /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     2056..2075
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2140..2159
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2270..2858
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2759..2778
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3012..3029
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3044..3183
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3109..3128)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3269..3291
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3285..3476)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4285..5367)
                     /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     5329..5348
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5368..5445)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5651..5670
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5754..5773
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5754..5772
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5773..5797
                     /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             5812..5834
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5853..5870
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5871..5891
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
ORIGIN
        1 aaaaaaacac taaatctgtg gtcttgaagc tcaatggacc catcgcttat ctgccccccg
       61 acttcttcca cagcaaaggt acagatctca agtacctggc gacagttgat atatactctt
      121 tgggcctgat gatactcgag attgcaacac agcaggaaat caagggcata catggtgttc
      181 tgataaagtc aatcgaggaa aactggcggg aagagtccca aatcacacgc ctctatacct
      241 ctcttggggc tgatgagctg aggcaggtga agatgtgtat tgatatagga cttgactgtg
      301 tcaagagtaa cccagagaag cgccccactg caggagcaat tatgttgtgg cttgataaag
      361 aatctaagcc agtccccgtg tcacgggccg gagcgggcgt tttgccacgg cccccagttc
      421 cgaccaacat caaccatgcg gggcgcatcc agggtatccc aatcgagaaa tcacaagtcc
      481 ttattccttc attcgcttgt aatgaatata gctctgtcaa aattcaaaat ttgcacttgc
      541 tgctctttat taacattgga agtggataac ggatccgaat tcgagcgccg tcgacaagct
      601 tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
      661 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
      721 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
      781 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
      841 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
      901 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
      961 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1021 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     1081 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     1141 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     1201 tttaacgcga attttaacaa actagtaacg tttacaattt caggtggcac ttttcgggga
     1261 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1321 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     1381 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     1441 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     1501 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     1561 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     1621 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     1681 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     1741 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tgttttcacc
     1801 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     1861 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     1921 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     1981 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     2041 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     2101 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     2161 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     2221 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2281 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2341 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2401 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2461 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2521 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2581 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2641 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2701 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2761 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2821 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2881 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2941 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3001 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3061 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     3121 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     3181 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3241 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3301 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3361 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     3421 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3481 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     3541 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     3601 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     3661 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     3721 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     3781 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     3841 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     3901 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     3961 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     4021 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     4081 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     4141 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     4201 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4261 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4321 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4381 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4441 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4501 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     4561 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     4621 catctgatcg ttggcaacca gcatcgcagt gggaacgata ccctcattca gcatttgcat
     4681 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     4741 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4801 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4861 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     4921 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     4981 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5041 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     5101 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     5161 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5221 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5281 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5341 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5401 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     5461 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     5521 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     5581 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     5641 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     5701 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     5761 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     5821 ctttaagaag gagatatacc atgggttctt ctcaccatca ccatcaccat gaaaacctgt
     5881 acttccaatc caatgccatg gggggatatg aattccagag agcagaattg gacgcccttg
     5941 agggtgtagt tcgtgaccca accgccgaac ccatgtcttt aacactacct cttttgcgtc
     6001 acattacgaa tgatttctcc cctgagttcg agatatctaa ggacgactcc gccgtggtat
     6061 atctaggagt gctaccatca ggatttaggg tggcagtgaa gaaatcacat tttaggtttt
     6121 gtctggacga cgaggacgcc ttcactaatg aagtttcaat tgcaatgaaa gccgcacata
     6181 agaatactgt aagagtaatc ggctattgtc atcatacaca tgagcagatt gccgagtatg
     6241 agggaaagca agtcttcgca gaagtgcgtg agaggttgat atgtactgag tacgtgccca
     6301 atggacctct atccggccat atcgaaggca aaatttgtgc acaaatggac ggctacgagt
     6361 tccagcgtgc tgagcttgat gcattggaga gagttgttag agatacctct gccgagccaa
     6421 tgtccttgac actgcccctt ttgcgtcata tcacaaatga tttctcagat gaatccagga
     6481 tcggacgtgg aggattcgct gttgtctatc ttggtgtttt gccatccggc ctgagaatag
     6541 ctgttaagag gttgtcaaat atagcttata tgaatgaaag tgcattccag aacgaagtat
     6601 tcatcacaat gaaagccaca cataaaaata cagtgagatt catgggatac tgctcacaga
     6661 ttcaaggcaa actaatagaa catgatggac aacatgtctt tgcacagctt gaagagaggc
     6721 taatttgtgt ggaatacgca cctaagggaa cattagacgc tcacatcgga gattatggtg
     6781 aactggactg gaaccaaaga tatcagattc tgaaaggcat atgtcagggt ttacatcatc
     6841 tacatgacga aatgcatgtc tttcatggag atatcaaacc cgccaacatt cttatcggcg
     6901 ataacctagt tcctaaaatc tatgatttcg gattgagtca aatgttcgag gaggaggaaa
     6961 cggaaagaat tgtggaaaac attgccggta cattcggcta catggcacct gagttctgta
     7021 caaacaacat ggtatccttc aaggccgaga tttacagttt aggcgttgta ataggagaac
     7081 ttcttatcgg taaaaaagga tggtttgacg aggacgtgcg gaagcttttt gttcagcagt
     7141 tgaaaggcct tcgcaaaact ttggtgaagg aaggcgcgtt ttccagctgg gagaataaat
     7201 accatcaagt gcgcacgtgt atggagattg gtcaggattg catcgatcca aatcctcata
     7261 aacggcctac gcttctcgaa attattcaac gccttaacga agccgaagac atgaattata
     7321 gtgcggcatc actctggcag gttcagtctg gtgatgaaga atcagatctc tccgatactg
     7381 aagcgttgga gacagagacc acgagcgagt ttcttccgtc cgatgaagaa ccggcgtccg
     7441 tcggtaagac cggcgagacc tctacacagg aaccggacaa acccgatctt atatctaaac
     7501 ttcccgcttc ggttgacctt tctgatctca aggtgcttga aaagataacc gatgatttta
     7561 gccatgagag aatagtcggg aaagacggga cttttaaagg atgccataag gctttcgttt
     7621 ataaaggtga cattcctctg agggagatga ttgcagttaa acggctcatt ggtgttgaga
     7681 tacccttcga gaagttcaag agagaggctg aacaattcat cagtttggat cataagaaca
     7741 tagttaaggt tgcaagttat tgccacgacc agagccgggg acatagactc gtccagttca
     7801 aggggaagcc tctgccccaa ctttttaatg ggcctgagca actgttgtgt tacgagtaca
     7861 tgcacaatgg ttctcttaga gattacttga tgggccaagg ctcccgggtc atagattggc
     7921 agatgcggta caagctgata aaagggacct gcgcaggctt gcattatctt cataagggtc
     7981 gcgcaggatg tccgatcgtt caccttaatc tgagtcccag caacgtgctt ttggatcata
     8041 attacattcc gcggataact ggcttcgact tttccaagct tataggag
//
