LOCUS       sequence-320904-        2529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TEV protease plasmid for T7-driven E. coli cell-free protein 
            synthesis with detection via HiBiT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2529)
  AUTHORS   Dudley QM, Cai YM, Kallam K, Debreyne H, Carrasco Lopez JA, Patron 
            NJ
  TITLE     Biofoundry-assisted expression and characterization of plant 
            proteins.
  JOURNAL   Synth Biol (Oxf). 2021 Sep 11;6(1):ysab029. doi: 
            10.1093/synbio/ysab029. eCollection 2021.
  PUBMED    34693026
REFERENCE   2  (bases 1 to 2529)
  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: "Synth Biol 
            (Oxf)."; date: "2021-09-11"; volume: "6(1):ysab029. doi"; pages: " 
            10.1093/synbio/ysab029. eCollection 2021"
COMMENT     Sequence Label: pEPQDKN0329
FEATURES             Location/Qualifiers
     source          1..2529
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      45..633
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     534..553
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     661..680
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        661..679
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             711..733
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             759..1466
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     primer_bind     complement(1558..1576)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1572..1619
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1637..2452)
                     /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="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2360..2379
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2444..2463
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 aacatcagag attttgagac acaacgtgag atcaaaggat cttcttgaga tccttttttt
       61 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
      121 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
      181 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
      241 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
      301 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
      361 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
      421 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
      481 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
      541 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
      601 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcgat cccgcgaaat
      661 taatacgact cactataggg acaccacaac ggtttccctc tagaaataat tttgtttaac
      721 tttaagaagg agatatacat atggagaaaa aaataatggg tgagtctctg ttcaaaggcc
      781 cacgcgacta taatcccatc tcctcgacga tctgccactt aaccaacgag tcggacggac
      841 atacgaccag tctgtacggc atcgggttcg gcccgtttat tatcactaat aaacatctgt
      901 tccgtcgcaa caacggtacc ttacttgtac agtctcttca cggcgtgttt aaagtgaaga
      961 atactactac ccttcagcag catctgatcg acggtcgcga tatgattatc atccgtatgc
     1021 cgaaagactt tccgcccttc ccgcagaaat taaagttccg cgaaccccag cgcgaggaac
     1081 gtatttgctt ggttaccaca aattttcaga caaaatcaat gagctcaatg gttagcgata
     1141 cgagctgtac ttttccgagc agtgacggta tcttttggaa acactggatc cagacgaaag
     1201 acggccaatg cggatcaccg ctggtgagta cccgtgacgg ctttatcgtg ggcattcatt
     1261 ccgccagcaa ctttactaat accaataact actttacctc agtacccaag aattttatgg
     1321 agctgttaac caaccaagaa gcacaacaat gggtgagcgg gtggcgtctg aacgccgata
     1381 gcgttctgtg gggtggtcac aaggtgttta tggtcaagcc tgaggaaccg ttccaacctg
     1441 tgaaagaggc cacccagttg atgaacggtt cggtgagcgg ctggcggctg ttcaagaaga
     1501 ttagctgagc ttcggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     1561 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     1621 tgaaagccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     1681 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     1741 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     1801 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     1861 caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga
     1921 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     1981 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     2041 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2101 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2161 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2221 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2281 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     2341 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2401 tggaatttaa tcgcggcttc gagcaagacg tttcccgttg aatatggctc ataacacccc
     2461 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt
     2521 gtgcaatgt
//
