LOCUS       sequence-270112-        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"
     CDS             complement(join(38..829,830..898))
                     /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     661..680
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(899..1003)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1071..1089
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1113..1127,1128..1133,1134..1150,1151..1157)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1133..1149
                     /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    1165..1181
                     /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             1200..1211
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1224..1244
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             1251..2348
                     /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      2205..2214
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2322..2345
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2397..2408
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2442..3149
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGTFWKHWIQTKDGQCGNPLVSTRDGFI
                     VGIHSASNFTNTNNYFASVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     terminator      3349..3443
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             3487..4146
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3553..3572)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      4211..4297
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4390..5472)
                     /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     5434..5453
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5473..5550)
                     /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(5634..5656)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5742..5882
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5797..5816
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5897..5914)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6068..6656)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6148..6167)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
       61 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
      121 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
      181 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
      241 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
      301 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
      361 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
      421 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
      481 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
      541 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
      601 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
      661 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
      721 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
      781 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
      841 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
      901 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
      961 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     1021 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     1081 tcacgaggcc ctttcgtctt cacctcgagt aatcataaaa aatttatttg ctttgtgagc
     1141 ggataacaat tataatagat tcaattgtga gcggataaca atttcacaca gaattcatta
     1201 aagaggagaa attaactatg aaacatcacc atcaccatca ccatggcggg aaaatcgaag
     1261 aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg
     1321 gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg
     1381 aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac
     1441 acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag
     1501 cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga
     1561 ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga
     1621 acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta
     1681 agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg
     1741 acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg
     1801 ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa aacaaacaca
     1861 tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga
     1921 tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg
     1981 taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg
     2041 caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc gaaaactatc
     2101 tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc
     2161 tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccacc atggaaaacg
     2221 cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc
     2281 gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg
     2341 cgcagactaa ttcgagctcg aacaacaaca acaataacaa taacaacaac ctcgggatcg
     2401 agggaagggg aagcggaggc ggagggtcga ccggtaccgg gggagaaagc ttgtttaagg
     2461 ggccgcgtga ttacaacccg atatcgagca ccatttgtca tttgacgaat gaatctgatg
     2521 ggcacacaac atcgttgtat ggtattggat ttggtccctt catcattaca aacaagcact
     2581 tgtttagaag aaataatgga acactgttgg tccaatcact acatggtgta ttcaaggtca
     2641 agaacaccac gactttgcaa caacacctca ttgatgggag ggacatgata attattcgca
     2701 tgcctaagga tttcccacca tttcctcaaa agctgaaatt tagagagcca caaagggaag
     2761 agcgcatatg tcttgtgaca accaacttcc aaactaagag catgtctagc atggtgtcag
     2821 acactagttg cacattccct tcatctgatg gcacattctg gaagcattgg attcaaacca
     2881 aggatgggca gtgtggcaat ccattagtat caactagaga tgggttcatt gttggtatac
     2941 actcagcatc gaatttcacc aacacaaaca attatttcgc aagcgtgccg aaaaacttca
     3001 tggaattgtt gacaaatcag gaggcgcagc agtgggttag tggttggcga ttaaatgctg
     3061 actcagtatt gtgggggggc cataaagttt tcatggtgaa acctgaagag ccttttcagc
     3121 cagttaagga agcgactcaa ctcatgaatt aataaggcgc gccttacccg cccatgcggg
     3181 caactttctc ttcgatttgc cggatttttt ccggcatttc ttgcgcaatg tggcgaaacg
     3241 cctcgctgat acgcggatct ctggcgaccg tttgccagaa ctgctgcgcc agcgggatcg
     3301 ccgttgggga tccgtcgacc tgcagccaag cttaattagc tgagcttgga ctcctgttga
     3361 tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc
     3421 gggcgttttt tattggtgag aatccaagct agcttggcga gattttcagg agctaaggaa
     3481 gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt
     3541 aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag
     3601 ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc
     3661 tttattcaca ttcttgcccg cctgatgaat gctcatccgg aatttcgtat ggcaatgaaa
     3721 gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa
     3781 actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac
     3841 atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt
     3901 attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta
     3961 aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg
     4021 caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc
     4081 ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg
     4141 gcgtaatttt tttaaggcag ttattggtgc ccttaaacgc ctggggtaat gactctctag
     4201 cttgaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     4261 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccctctagat tacgtgcagt
     4321 cgatgataag ctgtcaaaca tgagaattgt gcctaatgag tgagctaact tacattaatt
     4381 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4441 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     4501 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     4561 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     4621 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     4681 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     4741 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     4801 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     4861 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     4921 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     4981 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     5041 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     5101 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     5161 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5221 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     5281 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     5341 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     5401 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     5461 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     5521 gcgaaaggtt ttgcaccatt cgatggtttc ggaatttcgg gcagcgttgg gtcctggcca
     5581 cgggtgcgca tgatctagag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     5641 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     5701 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     5761 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     5821 gagtgcacca catgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     5881 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5941 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6001 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6061 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6121 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6181 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6241 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6301 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6361 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6421 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6481 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     6541 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6601 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6661 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6721 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6781 gttttaaat
//
