LOCUS       sequence-243597-        6533 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  His-tagged TEV protease with solubility increasing mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6533)
  AUTHORS   van den Berg S, Lofdahl PA, Hard T, Berglund H
  TITLE     Improved solubility of TEV protease by directed evolution.
  JOURNAL   J Biotechnol. 2006 Feb 10;121(3):291-8. doi: 
            10.1016/j.jbiotec.2005.08.006. Epub 2005 Sep 15.
  PUBMED    16150509
REFERENCE   2  (bases 1 to 6533)
  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"; doi: 
            "10.1016/j.jbiotec.2005.08.006"; journalName: "J Biotechnol"; date: 
            "2006-02-10- 10"; volume: "121"; issue: "3"; pages: "291-8"
COMMENT     Sequence Label: TEVSH
FEATURES             Location/Qualifiers
     source          1..6533
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..119
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        100..118
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    119..143
                     /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             158..180
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    200..224
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             227..934
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSSICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKDTTTLQQHLVDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMNKPE
                     EPFQPVKEATQLMN"
     protein_bind    complement(954..978)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1004..1045
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1055..1072
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1129..1147)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1143..1190
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1365..1370,1371..1387,1388..1393))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1424..1442)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1510..1614
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1615..1683,1684..2475)
                     /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(1833..1852)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2646..3234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3135..3154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3388..3405
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3420..3559
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3485..3504)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3645..3667
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3661..3852)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5164..6246)
                     /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     6208..6227
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6247..6324)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6530..6533,1..16)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
       61 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
      121 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
      181 tatacatatg cgttcatcaa caagtttgta caaaaaagca ggctcgggag aaagcttgtt
      241 taagggacca cgtgattata acccgatatc gagctccatt tgtcatttga cgaatgaatc
      301 tgatgggcac acaacatcgt tgtatggtat tggatttggt cccttcatca ttacaaacaa
      361 gcacttgttt agaagaaata atggaacact gttggtccaa tcactacatg gtgtattcaa
      421 ggtcaaggac accacgactt tgcaacaaca cctcgttgat gggagggaca tgataattat
      481 tcgcatgcct aaggatttcc caccatttcc tcaaaagctg aaatttagag agccacaaag
      541 ggaagagcgc atatgtcttg tgacaaccaa cttccaaact aagagtatgt ctagcatggt
      601 gtcagacact agttgcacat tcccttcatc tgatggcata ttctggaagc attggattca
      661 aaccaaggat gggcagtgtg gcagtccatt agtatcaact agagatgggt tcattgttgg
      721 tatacactca gcatcgaatt tcaccaacac aaacaattat ttcacaagcg tgccgaaaaa
      781 cttcatggaa ttgttgacaa atcaggaggc gcagcagtgg gttagtggtt ggcgattaaa
      841 tgctgactca gtattgtggg ggggccataa agttttcatg aacaaacctg aagagccttt
      901 tcagccagtt aaggaagcga ctcaactcat gaatgaattg gtgtactcgc aagacccagc
      961 tttcttgtac aaagtggttg atctggaggg cccgcggttc gaaggtaagc ctatccctaa
     1021 ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat caccatcacc attgagtttg
     1081 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     1141 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     1201 gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc
     1261 tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca
     1321 cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat
     1381 gataagctgt caaacatgag aattaattct tgaagacgaa agggcctcgt gatacgccta
     1441 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     1501 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1561 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     1621 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     1681 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1741 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1801 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     1861 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1921 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1981 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2041 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2101 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     2161 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2221 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2281 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     2341 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2401 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2461 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     2521 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2581 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2641 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2701 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2761 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     2821 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2881 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2941 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3001 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3061 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3121 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3181 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3241 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3301 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3361 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3421 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     3481 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     3541 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3601 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3661 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     3721 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     3781 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     3841 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     3901 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     3961 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     4021 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4081 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4141 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4201 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4261 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4321 ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat
     4381 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc
     4441 tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag
     4501 gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg
     4561 cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg
     4621 atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt
     4681 ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg
     4741 ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc
     4801 agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg
     4861 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4921 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     4981 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     5041 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     5101 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     5161 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5221 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     5281 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     5341 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     5401 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     5461 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5521 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5581 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5641 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5701 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5761 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5821 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5881 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5941 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     6001 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     6061 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     6121 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     6181 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     6241 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     6301 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     6361 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     6421 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     6481 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtg
//
