LOCUS       sequence-268950-        7354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of uTEV1-Delta protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7354)
  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 7354)
  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: MBP-TEVcs(ENLYFQ/G)-His6-uTEV1-Delta(220-242)-R5
FEATURES             Location/Qualifiers
     source          1..7354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    8..24
                     /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     13..35
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             94..1194
                     /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="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1051..1060
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1168..1191
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1243..1254
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1258..1278
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1279..1299
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     primer_bind     complement(2013..2030)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2013..2029)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2022..2044)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2208..2225)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2378..2464
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2556..2583
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2602..2693
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2694..2762,2763..3554)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2912..2931)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3596..4109)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3791..3812)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4003..4022
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4220..4808
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4709..4728
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4962..4979
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4994..5134
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5060..5079)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5220..5242
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5236..5427)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5923..6000
                     /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."
     CDS             6001..7083
                     /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(6020..6039)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(7326..7331,7332..7347,7348..7354)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 tgtggaattg tgagcggata acaatttcac acaggaaaca gccagtccgt ttaggtgttt
       61 tcacgagcac ttcaccaaca aggaccatag attatgaaaa ctgaagaagg taaactggta
      121 atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag
      181 aaagataccg gaattaaagt caccgttgag catccggata aactggaaga gaaattccca
      241 caggttgcgg caactggcga tggccctgac attatcttct gggcacacga ccgctttggt
      301 ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag
      361 ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc
      421 gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc
      481 tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg
      541 ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
      601 ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg
      661 aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc
      721 gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc
      781 ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg
      841 accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc
      901 gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa
      961 ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag
     1021 gaagagttgg cgaaagatcc acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa
     1081 atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc
     1141 aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactaattcg
     1201 agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgaggg aaggggagaa
     1261 aatctttatt ttcaaggtca tcatcatcat catcatcatg gagaaagctt gtttaagggg
     1321 ccgcgtgatt acaacccgat atcgagcacc atttgtcatt tgacgaatga atctgatggg
     1381 cacacaacat cgttgtatgg tattggattt ggtcccttca tcattacaaa caagcacttg
     1441 tttagaagaa ataatggaac actgttggtc caatcactac atggtgtatt caaggtcaag
     1501 aacaccacga ctttgcaaca acacctcatt gatgggaggg acatgataat tattcgcatg
     1561 cctaaggatt tcccaccatt tcctcaaaag ctgaaattta gagagccaca aagggaagag
     1621 cgcatatgtc ttgtgacaac caacttccaa actaagagca tgtctagcat ggtgtcagac
     1681 actagttgca cattcccttc atctgatggc atattctgga agcattggat tcaaaccaag
     1741 gatgggcagt gtggcaatcc attagtatca actagagatg ggttcattgt tggtatacac
     1801 tcagcatcga atttcaccaa cacaaacaat tatttcacaa gcgtgccgaa aaacttcatg
     1861 gaattgttga caaatcagga ggcgcagcag tgggttagtg gttggcgatt aaatgctgac
     1921 tcagtattgt gggggggcca taaagttttc atggtgcgtc gtcgccgtcg ctaataagga
     1981 tcctctagag tcgacctgca ggcaagcttg gcactggccg tcgttttaca acgtcgtgac
     2041 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     2101 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2161 ggcgaatggc agcttggctg ttttggcgga tgagataaga ttttcagcct gatacagatt
     2221 aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg
     2281 gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg
     2341 gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
     2401 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     2461 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
     2521 acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct
     2581 ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc
     2641 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2701 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2761 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2821 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2881 gtttcccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     2941 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     3001 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3061 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3121 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3181 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3241 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3301 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3361 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3421 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3481 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3541 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttacccc
     3601 ggttgataat cagaaaagcc ccaaaaacag gaagattgta taagcaaata tttaaattgt
     3661 aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3721 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagcccg agatagggtt
     3781 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3841 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac ccaaatcaag
     3901 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3961 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     4021 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     4081 cgcgcttaat gcgccgctac agggcgcgta aaaggatcta ggtgaagatc ctttttgata
     4141 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4201 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4261 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4321 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4381 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4441 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4501 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4561 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4621 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4681 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4741 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4801 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4861 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4921 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4981 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     5041 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     5101 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     5161 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     5221 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     5281 gtaaagctca tcagcgtggt cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc
     5341 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5401 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5461 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5521 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5581 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5641 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5701 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5761 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5821 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5881 catgcgcacc cgtggccagg acccaacgct gcccgaaatt ccgacaccat cgaatggtgc
     5941 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     6001 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     6061 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     6121 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     6181 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     6241 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     6301 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     6361 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     6421 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     6481 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     6541 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     6601 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     6661 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     6721 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     6781 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     6841 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     6901 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     6961 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     7021 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     7081 tgagcgcaac gcaattaatg taagttagct cactcattag gcacaattct catgtttgac
     7141 agcttatcat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct
     7201 gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc
     7261 cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg
     7321 agctgttgac aattaatcat cggctcgtat aatg
//
