LOCUS       sequence-471099-        6849 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 6849)
  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 6849)
  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:GST-VirPWT4
FEATURES             Location/Qualifiers
     source          1..6849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(121..139)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      135..182
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(699..717)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        785..889
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(890..958,959..1750)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1108..1127)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1921..2509
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2410..2429
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2663..2680
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2695..2834
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2760..2779)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2920..2942
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2936..3127)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5482..5501
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5521..5598)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5804..5823
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5907..5926
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5907..5925
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5926..5950
                     /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             5965..5987
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5994..6647
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             6654..6671
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             6678..6698
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
ORIGIN
        1 aaaaaaaaac tcaagattcg gaagggtcaa aaactcccac cacgcaaccg gaggatgagt
       61 gtggcctcga ggatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
      121 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
      181 tgctgaaagg aggaactata tccggatatc ccgcaagagg cccggcagta ccggcataac
      241 caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt
      301 agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta
      361 aagcttatcg atggccgccc aggccccagg ccttaagatc gtcatcggag gtagcagcac
      421 cctcgacggc ctcggcctta gaggcagcag cctcgacgtg cctcttgcaa tactcgatca
      481 ccttggcgag gatcttgctc gtgacgttag gaagagggac tccgttgtcg acgcagtcgt
      541 cttcaaccat atgcgctatg gtttgtgact cgagagccac cgcctcctca acctcgaaag
      601 attcaccatc ggaactcttc aacacaatct tcttcgcaga ggccagcgcg gccatcgatg
      661 ataagctgtc aaacatgaga attcttgaag acgaaagggc ctcgtgatac gcctattttt
      721 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
      781 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
      841 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
      901 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
      961 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1021 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1081 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     1141 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1201 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1261 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     1321 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     1381 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     1441 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     1501 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     1561 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggctctc gcggtatcat
     1621 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     1681 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     1741 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     1801 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     1861 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1921 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1981 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2041 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     2101 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2161 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2221 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2281 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2341 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2401 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2461 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2521 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2581 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     2641 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     2701 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     2761 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     2821 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2881 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     2941 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3001 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3061 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3121 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3181 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3241 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     3301 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     3361 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     3421 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     3481 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     3541 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     3601 acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata
     3661 tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa
     3721 ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc
     3781 ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac
     3841 aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag
     3901 cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg
     3961 atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc
     4021 ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa
     4081 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     4141 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     4201 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     4261 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     4321 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     4381 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     4441 actgcccgct ttccagtcgg gaaactgtcg tgccagctgc attaatgaat cggccaacgc
     4501 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     4561 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     4621 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     4681 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     4741 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     4801 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     4861 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4921 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4981 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     5041 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     5101 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     5161 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     5221 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     5281 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     5341 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     5401 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     5461 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     5521 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     5581 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     5641 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     5701 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     5761 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     5821 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     5881 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     5941 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgtccc
     6001 ctatactagg ttattggaaa attaagggcc ttgtgcaacc cactcgactt cttttggaat
     6061 atcttgaaga aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa
     6121 acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc ttattatatt gatggtgatg
     6181 ttaaattaac acagtctatg gccatcatac gttatatagc tgacaagcac aacatgttgg
     6241 gtggttgtcc aaaagagcgt gcagagattt caatgcttga aggagcggtt ttggatatta
     6301 gatacggtgt ttcgagaatt gcatatagta aagactttga aactctcaaa gttgattttc
     6361 ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt
     6421 taaatggtga tcatgtaacc catcctgact tcatgttgta tgacgctctt gatgttgttt
     6481 tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta
     6541 ttgaagctat cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt
     6601 tgcagggctg gcaagccacg tttggtggtg gcgaccatcc tccaaaatcg gatctggttc
     6661 cgcgtggatc tcatagcgaa aacctctact tccaatcgat gctgaattcc ccagaagtcc
     6721 cgacttataa aattggacat cctgggcctc ggtatattag atataacgat ggtgaaatcc
     6781 atgaggtcga acgcaaggaa tcagggcgct atcgagtaga cgacgggggt ttaatacgtg
     6841 aggaatatg
//
