LOCUS       sequence-443586-        6407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His-tagged mutant (L56V/S135G/S219V) TEV protease in E. 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6407)
  AUTHORS   Parida PP, Saraswathi D, Mopidevi SMV, Raran-Kurussi S
  TITLE     Advancing large-scale production of TEV protease through an 
            innovative NT* tag-based fusion construct.
  JOURNAL   Curr Res Struct Biol. 2023 Sep 30;6:100106. doi: 
            10.1016/j.crstbi.2023.100106. eCollection 2023.
  PUBMED    37822550
REFERENCE   2  (bases 1 to 6407)
  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: "Curr Res 
            Struct Biol."; date: "2023-09-30"; volume: "6:100106. doi"; pages: "
            10.1016/j.crstbi.2023.100106. eCollection 2023"
COMMENT     Sequence Label: pSRK51
FEATURES             Location/Qualifiers
     source          1..6407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(91..112)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     303..322
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(446..493)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     489..507
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(560..577)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    590..614
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(619..1326)
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLVVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSGDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     CDS             complement(1327..1347)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             complement(1348..1368)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(1368..1392)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     RBS             1768..1790
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1805..1829
                     /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)."
     primer_bind     complement(1829..1848)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1830..1848)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1932..1951)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2157..2234
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2235..3317
                     /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     complement(2254..2273)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4126..4317
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4311..4333)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4419..4558
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4474..4493
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4573..4590)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4744..5332)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4824..4843)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5443..5462)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5454..6269
                     /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             5454..6269
                     /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     complement(5527..5546)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(6361..6407,1..409))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
       61 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
      121 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
      181 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
      241 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
      301 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
      361 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
      421 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
      481 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      541 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtacatca ccactttgta
      601 caagaaagtt gattattaat tcatgagttg agtcgcttcc ttaactggct gaaaaggctc
      661 ttcaggtttc accatgaaaa ctttatggcc cccccacaat actgagtcag catttaatcg
      721 ccaaccacta acccactgct gcgcctcctg atttgtcaac aattccatga agtttttcgg
      781 cacgcttgtg aaataattgt ttgtgttggt gaaattcgat gctgagtgta taccaacaat
      841 gaacccatct ctagttgata ctaatggact gccacactgc ccatccttgg tttgaatcca
      901 atgcttccag aatatgccat cacctgaagg gaatgtgcaa ctagtgtctg acaccatgct
      961 agacatgctc ttagtttgga agttggttgt cacaagacat atgcgctctt ccctttgtgg
     1021 ctctctaaat ttcagctttt gaggaaatgg tgggaaatcc ttaggcatgc gaataattat
     1081 catgtccctc ccatcaatga ggtgttgttg caaagtcgtg gtgttcttga ccttgaatac
     1141 accatgtagt gattggacca ccagtgttcc attatttctt ctaaacaagt gcttgtttgt
     1201 aatgatgaag ggaccaaatc caataccata caacgatgtt gtgtgcccat cagattcatt
     1261 cgtcaaatga caaatggtgc tcgatatcgg gttgtaatca cgcggcccct taaacaagct
     1321 ttctccatga tgatgatgat gatgatgacc ttggaagtac aggttttcgc caactttttt
     1381 gtacaaactt gtgatcaact gcgtgatctc gttaataaag ggctggttga ccacgccagt
     1441 ggtctgtaaa aacgcattgc tcatcgccga ggcaatcgag ctggttttcg tagaaagaga
     1501 cccgccgcct tcctcactgg ctgcaatttc tgccatgcta ctggcgaaag ccatgttcag
     1561 ggcctgcaaa tcattggggg aagtacgccc ttgggccgcc aggctttgaa tcgactgaac
     1621 cattgattga gcaatggtgg acatcttgtc aagttggctc gctgtgaatc caggcataga
     1681 ggacaggccc tgcatgaatg aattcataaa attctccgct aagccaggat tggtccaagg
     1741 agttgtgtga ctcatgccca tggtatatct ccttcttaaa gttaaacaaa attatttcta
     1801 gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat
     1861 cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
     1921 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     1981 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     2041 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     2101 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     2161 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     2221 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     2281 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     2341 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     2401 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     2461 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     2521 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     2581 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     2641 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     2701 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     2761 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     2821 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     2881 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     2941 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     3001 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     3061 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     3121 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     3181 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     3241 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3301 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg
     3361 ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg
     3421 ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag
     3481 gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg
     3541 atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact
     3601 ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca
     3661 cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt
     3721 actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa
     3781 acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
     3841 gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc
     3901 tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg
     3961 atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt
     4021 aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt
     4081 atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
     4141 aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa
     4201 ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct
     4261 gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     4321 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     4381 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     4441 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     4501 tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     4561 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4621 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4681 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4741 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4801 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4861 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4921 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4981 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5041 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5101 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5161 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     5221 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5281 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5341 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5401 ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     5461 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
     5521 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
     5581 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     5641 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     5701 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     5761 ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     5821 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     5881 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     5941 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     6001 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     6061 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     6121 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     6181 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     6241 ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa
     6301 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6361 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgt
//
