LOCUS       sequence-471105-        6879 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 6879)
  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 6879)
  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-AvrPWT4
FEATURES             Location/Qualifiers
     source          1..6879
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(798..817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     958..980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(974..1165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3520..3539
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3559..3636)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3842..3861
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3945..3964
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3945..3963
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3964..3988
                     /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             4003..4025
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4032..4685
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             4692..4709
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4716..4736
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5038..5056)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5052..5099
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(5616..5634)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5702..5806
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5807..5875,5876..6667)
                     /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(6025..6044)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6838..6879,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     1201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     1321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     1381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     1441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     1501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     1561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     1621 tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga
     1681 tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca
     1741 agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc
     1801 cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag
     1861 gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata
     1921 aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga
     1981 tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc
     2041 gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg
     2101 cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc
     2161 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     2221 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     2281 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     2341 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     2401 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     2461 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aactgtcgtg ccagctgcat
     2521 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     2581 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     2641 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     2701 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     2761 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     2821 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     2881 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     2941 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     3001 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     3061 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     3121 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     3181 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     3241 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     3301 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     3361 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     3421 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     3481 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     3541 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     3601 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     3661 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     3721 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     3781 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     3841 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     3901 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     3961 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     4021 ggagatatac catgtcccct atactaggtt attggaaaat taagggcctt gtgcaaccca
     4081 ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg
     4141 aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt
     4201 attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg
     4261 acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag
     4321 gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa
     4381 ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt
     4441 tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg
     4501 acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag
     4561 tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca
     4621 agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc
     4681 caaaatcgga tctggttccg cgtggatctc atagcgaaaa cctctacttc caatcgatga
     4741 aacccccctc cgattcgacg gatgcctcaa aaatcccgac ttacaaaatt aaacatccag
     4801 ggcctcgtta cattataact gacgatggtc gagttgttga ggtcgaacgc aaggaatcag
     4861 gacgctatca attaatcgat ggggttttag tacgtgtgga atatgaaaag aaaaccgggg
     4921 gttcggaagg gtcaaaaaca tccaccaaca cgcgcggtcc gaaaccggag gatgagtgtg
     4981 gcctcgagga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     5041 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     5101 tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg gcataaccaa
     5161 gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga
     5221 tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag
     5281 cttatcgatg gccgcccagg ccccaggcct taagatcgtc atcggaggta gcagcaccct
     5341 cgacggcctc ggccttagag gcagcagcct cgacgtgcct cttgcaatac tcgatcacct
     5401 tggcgaggat cttgctcgtg acgttaggaa gagggactcc gttgtcgacg cagtcgtctt
     5461 caaccatatg cgctatggtt tgtgactcga gagccaccgc ctcctcaacc tcgaaagatt
     5521 caccatcgga actcttcaac acaatcttct tcgcagaggc cagcgcggcc atcgatgata
     5581 agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     5641 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5701 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5761 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5821 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5881 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5941 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     6001 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     6061 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6121 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6181 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6241 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6301 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     6361 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6421 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6481 tggctggttt attgctgata aatctggagc cggtgagcgt ggctctcgcg gtatcattgc
     6541 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6601 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6661 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6721 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6781 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6841 agatcctttt tttctgcgcg taatctgctg cttgcaaac
//
