LOCUS       sequence-445157-        7390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His-tagged mutant (C19V/L56V/C110V/C130S/S135G/S219V) TEV 
            protease in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7390)
  AUTHORS   Davis SM, Romig BL, Abe AA, Loening NM
  TITLE     An improved variant of tobacco etch virus (TEV) protease that does 
            not need reducing agents.
  JOURNAL   Protein Sci. 2025 Mar;34(3):e70049. doi: 10.1002/pro.70049.
  PUBMED    39969093
REFERENCE   2  (bases 1 to 7390)
  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: "Protein 
            Sci."; date: "2025-03"; volume: "34(3):e70049. doi"; pages: " 
            10.1002/pro.70049"
COMMENT     Sequence Label: pMAL-7xHis-TEV-hexa
FEATURES             Location/Qualifiers
     source          1..7390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..30
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    45..185
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(111..130)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     271..293
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(287..478)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        974..1051
                     /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             1052..2134
                     /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(1071..1090)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2377..2382,2383..2398,2399..2405)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2413..2429
                     /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     2418..2440
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             2499..3599
                     /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      3456..3465
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3573..3596
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3648..3659
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3663..3683
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3684..3704
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             3705..4412
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTIVHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLVVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERIVLVTTNFQTKSMSSMVSDTSSTFPSGDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     primer_bind     complement(4454..4471)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4454..4470)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4463..4485)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4649..4666)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4819..4905
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4997..5024
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5043..5134
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5135..5203,5204..5995)
                     /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(5353..5372)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6037..6550)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6232..6253)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6444..6463
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      6661..7249
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7150..7169
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
       61 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
      121 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
      181 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
      241 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
      301 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgcagcg
      361 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
      421 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
      481 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
      541 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
      601 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
      661 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
      721 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
      781 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
      841 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
      901 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc
      961 tgcccgaaat tccgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg
     1021 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     1081 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     1141 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     1201 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     1261 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     1321 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     1381 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     1441 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     1501 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     1561 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     1621 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     1681 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     1741 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     1801 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
     1861 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     1921 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     1981 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     2041 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2101 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     2161 tcactcatta ggcacaattc tcatgtttga cagcttatca tcgactgcac ggtgcaccaa
     2221 tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac
     2281 tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca
     2341 tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca tcggctcgta
     2401 taatgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagccag tccgtttagg
     2461 tgttttcacg agcacttcac caacaaggac catagattat gaaaactgaa gaaggtaaac
     2521 tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat
     2581 tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat
     2641 tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct
     2701 ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg
     2761 acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc
     2821 cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa
     2881 aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc
     2941 tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt
     3001 atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg
     3061 gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag
     3121 acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca
     3181 acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac
     3241 tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta
     3301 acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg
     3361 atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt
     3421 acgaggaaga gttggcgaaa gatccacgta ttgccgccac catggaaaac gcccagaaag
     3481 gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg
     3541 tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta
     3601 attcgagctc gaacaacaac aacaataaca ataacaacaa cctcgggatc gagggaaggg
     3661 gagaaaatct ttattttcaa ggtcatcatc atcatcatca tcatggagaa agcttgttta
     3721 aggggccgcg tgattacaac ccgatatcga gcaccattgt tcatttgacg aatgaatctg
     3781 atgggcacac aacatcgttg tatggtattg gatttggtcc cttcatcatt acaaacaagc
     3841 acttgtttag aagaaataat ggaacactgg tggtccaatc actacatggt gtattcaagg
     3901 tcaagaacac cacgactttg caacaacacc tcattgatgg gagggacatg ataattattc
     3961 gcatgcctaa ggatttccca ccatttcctc aaaagctgaa atttagagag ccacaaaggg
     4021 aagagcgcat agttcttgtg acaaccaact tccaaactaa gagcatgtct agcatggtgt
     4081 cagacactag tagcacattc ccttcaggtg atggcatatt ctggaagcat tggattcaaa
     4141 ccaaggatgg gcagtgtggc agtccattag tatcaactag agatgggttc attgttggta
     4201 tacactcagc atcgaatttc accaacacaa acaattattt cacaagcgtg ccgaaaaact
     4261 tcatggaatt gttgacaaat caggaggcgc agcagtgggt tagtggttgg cgattaaatg
     4321 ctgactcagt attgtggggg ggccataaag ttttcatggt gaaacctgaa gagccttttc
     4381 agccagttaa ggaagcgact caactcatga attaataagg atcctctaga gtcgacctgc
     4441 aggcaagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4501 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4561 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cagcttggct
     4621 gttttggcgg atgagataag attttcagcc tgatacagat taaatcagaa cgcagaagcg
     4681 gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc
     4741 cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag
     4801 tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     4861 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
     4921 ttgaacgttg cgaagcaacg gcccggaggg kggcgggcag gmcgcccgcc ataaactgcc
     4981 aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact
     5041 cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5101 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5161 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5221 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5281 caacagcggt aagatccttg agagttttcg ccccgaagaa cgtttcccaa tgatgagcac
     5341 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     5401 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5461 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5521 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5581 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5641 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5701 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5761 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5821 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5881 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5941 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6001 agaccaagtt tactcatata tactttagat tgatttaccc cggttgataa tcagaaaagc
     6061 cccaaaaaca ggaagattgt ataagcaaat atttaaattg taaacgttaa tattttgtta
     6121 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     6181 aaaatccctt ataaatcaaa agaatagccc gagatagggt tgagtgttgt tccagtttgg
     6241 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     6301 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc
     6361 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     6421 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     6481 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     6541 cagggcgcgt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6601 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6661 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6721 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6781 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     6841 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6901 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6961 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     7021 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     7081 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     7141 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     7201 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     7261 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     7321 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     7381 ccgcagccga
//
