LOCUS       sequence-261443-        6789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of uTEV3 (full-length) protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6789)
  AUTHORS   Sanchez MI, Ting AY
  TITLE     Directed evolution improves the catalytic efficiency of TEV 
            protease.
  JOURNAL   Nat Methods. 2019 Dec 9. pii: 10.1038/s41592-019-0665-7. doi: 
            10.1038/s41592-019-0665-7.
  PUBMED    31819267
REFERENCE   2  (bases 1 to 6789)
  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 
            Methods. 2019 Dec 9. pii: 10.1038/s41592-019-0665-7. doi: 
            10.1038/s41592-019-0665-7."
COMMENT     Sequence Label: His6-MBP-uTEV3
FEATURES             Location/Qualifiers
     source          1..6789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             121..141
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             148..1245
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1102..1111
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1219..1242
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1294..1305
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1339..2046
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGTFWKHWIQTKDGQCGNPLVSTRDGFI
                     VGIHSASNFTNTNNYFASVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     terminator      2246..2340
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2384..3043
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2450..2469)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3108..3194
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3287..4369)
                     /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     4331..4350
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4370..4447)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(4531..4553)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4639..4779
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4694..4713
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4794..4811)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4965..5553)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5045..5064)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5724..6515,6516..6584))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6347..6366
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6585..6689)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6757..6775
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaaa
      121 catcaccatc accatcacca tggcgggaaa atcgaagaag gtaaactggt aatctggatt
      181 aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc
      241 ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg
      301 gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct
      361 caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg
      421 tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa
      481 gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag
      541 atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg
      601 caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat
      661 gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt
      721 ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc
      781 atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca
      841 tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag
      901 ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg
      961 aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa
     1021 gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg
     1081 gcgaaagatc cacgtattgc cgccaccatg gaaaacgccc agaaaggtga aatcatgccg
     1141 aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc
     1201 agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaattc gagctcgaac
     1261 aacaacaaca ataacaataa caacaacctc gggatcgagg gaaggggaag cggaggcgga
     1321 gggtcgaccg gtaccggggg agaaagcttg tttaaggggc cgcgtgatta caacccgata
     1381 tcgagcacca tttgtcattt gacgaatgaa tctgatgggc acacaacatc gttgtatggt
     1441 attggatttg gtcccttcat cattacaaac aagcacttgt ttagaagaaa taatggaaca
     1501 ctgttggtcc aatcactaca tggtgtattc aaggtcaaga acaccacgac tttgcaacaa
     1561 cacctcattg atgggaggga catgataatt attcgcatgc ctaaggattt cccaccattt
     1621 cctcaaaagc tgaaatttag agagccacaa agggaagagc gcatatgtct tgtgacaacc
     1681 aacttccaaa ctaagagcat gtctagcatg gtgtcagaca ctagttgcac attcccttca
     1741 tctgatggca cattctggaa gcattggatt caaaccaagg atgggcagtg tggcaatcca
     1801 ttagtatcaa ctagagatgg gttcattgtt ggtatacact cagcatcgaa tttcaccaac
     1861 acaaacaatt atttcgcaag cgtgccgaaa aacttcatgg aattgttgac aaatcaggag
     1921 gcgcagcagt gggttagtgg ttggcgatta aatgctgact cagtattgtg ggggggccat
     1981 aaagttttca tggtgaaacc tgaagagcct tttcagccag ttaaggaagc gactcaactc
     2041 atgaattaat aaggcgcgcc ttacccgccc atgcgggcaa ctttctcttc gatttgccgg
     2101 attttttccg gcatttcttg cgcaatgtgg cgaaacgcct cgctgatacg cggatctctg
     2161 gcgaccgttt gccagaactg ctgcgccagc gggatcgccg ttggggatcc gtcgacctgc
     2221 agccaagctt aattagctga gcttggactc ctgttgatag atccagtaat gacctcagaa
     2281 ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat
     2341 ccaagctagc ttggcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac
     2401 tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca
     2461 gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa
     2521 gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct
     2581 gatgaatgct catccggaat ttcgtatggc aatgaaagac ggtgagctgg tgatatggga
     2641 tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg
     2701 gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg
     2761 ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc
     2821 agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt
     2881 cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc
     2941 gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa
     3001 tgaattacaa cagtactgcg atgagtggca gggcggggcg taattttttt aaggcagtta
     3061 ttggtgccct taaacgcctg gggtaatgac tctctagctt gaggcatcaa ataaaacgaa
     3121 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     3181 tgagtaggac aaatccgccc tctagattac gtgcagtcga tgataagctg tcaaacatga
     3241 gaattgtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     3301 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3361 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     3421 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     3481 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     3541 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     3601 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     3661 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     3721 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     3781 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     3841 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     3901 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     3961 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     4021 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     4081 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     4141 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     4201 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     4261 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     4321 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     4381 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga
     4441 tggtgtcgga atttcgggca gcgttgggtc ctggccacgg gtgcgcatga tctagagctg
     4501 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     4561 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     4621 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     4681 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccacat gcggtgtgaa
     4741 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     4801 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4861 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4921 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4981 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5041 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5101 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5161 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5221 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5281 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5341 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5401 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5461 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5521 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5581 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5641 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5701 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5761 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5821 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5881 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5941 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6001 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6061 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6121 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6181 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6241 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6301 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6361 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6421 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6481 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6541 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6601 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6661 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6721 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     6781 cgtcttcac
//
