LOCUS       sequence-268952-        7354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of TEV-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-TEV-Delta(220-242)-R5
FEATURES             Location/Qualifiers
     source          1..7354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             123..1223
                     /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      1080..1089
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1197..1220
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1272..1283
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1287..1307
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1308..1328
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     primer_bind     complement(2042..2059)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2042..2058)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2051..2073)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2237..2254)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2407..2493
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2585..2612
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2631..2722
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2723..2791,2792..3583)
                     /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(2941..2960)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3625..4138)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3820..3841)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4032..4051
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4249..4837
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4738..4757
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4991..5008
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5023..5163
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5089..5108)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5249..5271
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5265..5456)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5952..6029
                     /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             6030..7112
                     /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(6049..6068)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaac tgaagaaggt aaactggtaa tctggattaa cggcgataaa ggctataacg
      181 gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc accgttgagc
      241 atccggataa actggaagag aaattcccac aggttgcggc aactggcgat ggccctgaca
      301 ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg ttggctgaaa
      361 tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat gccgtacgtt
      421 acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg atttataaca
      481 aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg gataaagaac
      541 tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac ttcacctggc
      601 cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag tacgacatta
      661 aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg gttgacctga
      721 ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct gcctttaata
      781 aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc gacaccagca
      841 aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg
      901 ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg gcaaaagagt
      961 tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa gacaaaccgc
     1021 tgggtgccgt agcgctgaag tcttacgagg aagagttggc gaaagatcca cgtattgccg
     1081 ccaccatgga aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt
     1141 tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag actgtcgatg
     1201 aagccctgaa agacgcgcag actaattcga gctcgaacaa caacaacaat aacaataaca
     1261 acaacctcgg gatcgaggga aggggagaaa atctttattt tcaaggtcat catcatcatc
     1321 atcatcatgg agaaagcttg tttaaggggc cgcgtgatta caacccgata tcgagcacca
     1381 tttgtcattt gacgaatgaa tctgatgggc acacaacatc gttgtatggt attggatttg
     1441 gtcccttcat cattacaaac aagcacttgt ttagaagaaa taatggaaca ctgttggtcc
     1501 aatcactaca tggtgtattc aaggtcaaga acaccacgac tttgcaacaa cacctcattg
     1561 atgggaggga catgataatt attcgcatgc ctaaggattt cccaccattt cctcaaaagc
     1621 tgaaatttag agagccacaa agggaagagc gcatatgtct tgtgacaacc aacttccaaa
     1681 ctaagagcat gtctagcatg gtgtcagaca ctagttgcac attcccttca tctgatggca
     1741 tattctggaa gcattggatt caaaccaagg atgggcagtg tggcagtcca ttagtatcaa
     1801 ctagagatgg gttcattgtt ggtatacact cagcatcgaa tttcaccaac acaaacaatt
     1861 atttcacaag cgtgccgaaa aacttcatgg aattgttgac aaatcaggag gcgcagcagt
     1921 gggttagtgg ttggcgatta aatgctgact cagtattgtg ggggggccat aaagttttca
     1981 tggtgcgtcg tcgccgtcgc taataaggat cctctagagt cgacctgcag gcaagcttgg
     2041 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     2101 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     2161 gcccttccca acagttgcgc agcctgaatg gcgaatggca gcttggctgt tttggcggat
     2221 gagataagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     2281 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     2341 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     2401 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     2461 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     2521 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
     2581 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt
     2641 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     2701 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     2761 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     2821 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     2881 gatccttgag agttttcgcc ccgaagaacg tttcccaatg atgagcactt ttaaagttct
     2941 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     3001 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3061 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3121 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3181 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3241 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3301 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3361 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3421 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     3481 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3541 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     3601 ctcatatata ctttagattg atttaccccg gttgataatc agaaaagccc caaaaacagg
     3661 aagattgtat aagcaaatat ttaaattgta aacgttaata ttttgttaaa attcgcgtta
     3721 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3781 aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     3841 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     3901 ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg taaagcacta
     3961 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     4021 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     4081 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtaa
     4141 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4201 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4261 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4321 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4381 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4441 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4501 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4561 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4621 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4681 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4741 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4801 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4861 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4921 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4981 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     5041 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     5101 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     5161 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5221 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5281 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgcagcgat
     5341 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     5401 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     5461 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     5521 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     5581 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     5641 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     5701 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     5761 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     5821 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     5881 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg
     5941 cccgaaattc cgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc
     6001 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     6061 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     6121 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     6181 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     6241 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     6301 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     6361 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     6421 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     6481 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     6541 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     6601 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     6661 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     6721 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     6781 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     6841 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     6901 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     6961 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     7021 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     7081 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     7141 actcattagg cacaattctc atgtttgaca gcttatcatc gactgcacgg tgcaccaatg
     7201 cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg
     7261 cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc
     7321 ataacggttc tggcaaatat tctgaaatga gctg
//
