LOCUS       sequence-268951-        7354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of uTEV2-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-uTEV2-Delta(220-242)-R5
FEATURES             Location/Qualifiers
     source          1..7354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(192..197,198..213,214..220)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    228..244
                     /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     233..255
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             314..1414
                     /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      1271..1280
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1388..1411
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1463..1474
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1478..1498
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1499..1519
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     primer_bind     complement(2233..2250)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2233..2249)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2242..2264)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2428..2445)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2598..2684
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2776..2803
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2822..2913
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2914..2982,2983..3774)
                     /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(3132..3151)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3816..4329)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4011..4032)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4223..4242
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4440..5028
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4929..4948
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5182..5199
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5214..5354
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5280..5299)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5440..5462
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5456..5647)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6143..6220
                     /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             6221..7303
                     /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(6240..6259)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg
       61 gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg
      121 cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct
      181 gaaatgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata
      241 acaatttcac acaggaaaca gccagtccgt ttaggtgttt tcacgagcac ttcaccaaca
      301 aggaccatag attatgaaaa ctgaagaagg taaactggta atctggatta acggcgataa
      361 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt
      421 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga
      481 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct
      541 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga
      601 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct
      661 gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
      721 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta
      781 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa
      841 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
      901 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc
      961 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat
     1021 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
     1081 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct
     1141 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa
     1201 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
     1261 acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca
     1321 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca
     1381 gactgtcgat gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa
     1441 taacaataac aacaacctcg ggatcgaggg aaggggagaa aatctttatt ttcaaggtca
     1501 tcatcatcat catcatcatg gagaaagctt gtttaagggg ccgcgtgatt acaacccgat
     1561 atcgagcacc atttgtcatt tgacgaatga atctgatggg cacacagcat cgttgtatgg
     1621 tattggattt ggtcccttca tcattacaaa caagcacttg tttagaagaa ataatggaac
     1681 actgttggtc caatcactac atggtgtatt caaggtcaag aacaccacga ctttgcaaca
     1741 acacctcatt gatgggaggg acatgataat tattcgcatg cctaaggatt tcccaccatt
     1801 tcctcaaaag ctgaaattta gagagccaca aagggaagag cgcatatgtc ttgtgacaac
     1861 caacttccaa actaagagca tgtctagcat ggtgtcagac actagttgca cattcccttc
     1921 atctgatggc atattctgga agcattggat tcaaaccaag gatgggcagt gtggcaatcc
     1981 attagtatca actagagatg ggttcattgt tggtatacac tcagcatcga atttcaccaa
     2041 cacaaacaat tatttcacaa gcgtgccgaa aaacttcatg gaattgttga caaatcagga
     2101 ggcgcagcag tgggttagtg gttggcgatt aaatgctgac tcagtattgt gggggggcca
     2161 taaagttttc atggtgcgtc gtcgccgtcg ctaataagga tcctctagag tcgacctgca
     2221 ggcaagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2281 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2341 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc agcttggctg
     2401 ttttggcgga tgagataaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
     2461 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc
     2521 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt
     2581 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     2641 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     2701 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     2761 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
     2821 ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2881 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2941 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3001 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3061 aacagcggta agatccttga gagttttcgc cccgaagaac gtttcccaat gatgagcact
     3121 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     3181 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     3241 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     3301 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3361 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3421 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     3481 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3541 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3601 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3661 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3721 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3781 gaccaagttt actcatatat actttagatt gatttacccc ggttgataat cagaaaagcc
     3841 ccaaaaacag gaagattgta taagcaaata tttaaattgt aaacgttaat attttgttaa
     3901 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     3961 aaatccctta taaatcaaaa gaatagcccg agatagggtt gagtgttgtt ccagtttgga
     4021 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     4081 agggcgatgg cccactacgt gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc
     4141 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     4201 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     4261 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     4321 agggcgcgta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     4381 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4441 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4501 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4561 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4621 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4681 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4741 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4801 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4861 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4921 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4981 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5041 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     5101 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     5161 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     5221 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
     5281 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     5341 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5401 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5461 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     5521 cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     5581 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     5641 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     5701 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     5761 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     5821 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     5881 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     5941 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     6001 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     6061 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     6121 acccaacgct gcccgaaatt ccgacaccat cgaatggtgc aaaacctttc gcggtatggc
     6181 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     6241 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     6301 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     6361 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     6421 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     6481 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     6541 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     6601 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     6661 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     6721 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     6781 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     6841 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     6901 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     6961 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     7021 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     7081 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     7141 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     7201 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     7261 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     7321 taagttagct cactcattag gcacaattct catg
//
