LOCUS       sequence-443047-        7170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Maltose-binding protein-super TEV protease in bacterial 
            cytoplasm. This protease version performs well in the absence of 
            added reducing agent..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7170)
  AUTHORS   Keeble AH, Yadav VK, Ferla MP, Bauer CC, Chuntharpursat-Bon E, Huang
            J, Bon RS, Howarth M
  TITLE     DogCatcher allows loop-friendly protein-protein ligation.
  JOURNAL   Cell Chem Biol. 2021 Jul 28. pii: S2451-9456(21)00315-9. doi: 
            10.1016/j.chembiol.2021.07.005.
  PUBMED    34324879
REFERENCE   2  (bases 1 to 7170)
  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: "Cell Chem 
            Biol. 2021 Jul 28. pii: S2451-9456(21)00315-9. doi: 
            10.1016/j.chembiol.2021.07.005."
COMMENT     Sequence Label: pET28-MBP-super TEV protease
FEATURES             Location/Qualifiers
     source          1..7170
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      23..32
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     140..163
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             215..226
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             242..262
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             263..970
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTIVHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLVVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERIVLVTTNFQTKSMSSMVSDTSSTFPSGDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMDKPE
                     EPFQPVKEATQLMN"
     CDS             1013..1030
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1083..1101)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1097..1144
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1181..1636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1268..1287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1478..1499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1728..2543)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1728..2543)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2451..2470
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2535..2554
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2665..3253
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3154..3173
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3407..3424
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3439..3578
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3504..3523)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3664..3686
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3680..3871)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4680..5762)
                     /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     5724..5743
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5763..5840)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6046..6065
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6149..6168
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6149..6167
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6168..6192
                     /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             6207..6229
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(6236..7170,1..166)
                     /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="MGIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
ORIGIN
        1 ggcgaaagat ccacgtattg ccgccaccat ggaaaacgcc cagaaaggtg aaatcatgcc
       61 gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc
      121 cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt cgagctcgaa
      181 caacaacaac aataacaata acaacaacct cgggatcgag ggaaggggag attttgcagg
      241 tcatcatcat catcatcatc atggagaaag cttgtttaag gggccgcgtg attacaaccc
      301 gatatcgagc accattgttc atttgacgaa tgaatctgat gggcacacaa catcgttgta
      361 tggtattgga tttggtccct tcatcattac aaacaagcac ttgtttagaa gaaataatgg
      421 aacactggtg gtccaatcac tacatggtgt attcaaggtc aagaacacca cgactttgca
      481 acaacacctc attgatggga gggacatgat aattattcgc atgcctaagg atttcccacc
      541 atttcctcaa aagctgaaat ttagagagcc acaaagggaa gagcgcatag tccttgtgac
      601 aaccaacttc caaactaaga gcatgtctag catggtgtca gacactagtt cgacattccc
      661 ttcaggagat ggcatattct ggaagcattg gattcaaacc aaggatgggc agtgtggcag
      721 tccattagta tcaactagag atgggttcat tgttggtata cactcagcat cgaatttcac
      781 caacacaaac aattatttca caagcgtgcc gaaaaacttc atggaattgt tgacaaatca
      841 ggaggcgcag cagtgggtta gtggttggcg attaaatgct gactcagtat tgtggggagg
      901 ccataaagtt ttcatggaca aacctgaaga gccttttcag ccagttaagg aagcgactca
      961 actcatgaat cgtcgtcgcc gtcgctaata aaagcttgcg gccgcactcg agcaccacca
     1021 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     1081 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     1141 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     1201 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     1261 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     1321 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     1381 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     1441 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     1501 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     1561 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     1621 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1681 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     1741 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     1801 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     1861 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     1921 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     1981 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     2041 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     2101 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     2161 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     2221 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     2281 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     2341 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     2401 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     2461 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     2521 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     2581 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2641 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2701 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2761 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     2821 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2881 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2941 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3001 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3061 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3121 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3181 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3241 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3301 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3361 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3421 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3481 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3541 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     3601 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3661 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     3721 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     3781 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     3841 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     3901 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     3961 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     4021 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     4081 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     4141 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     4201 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     4261 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     4321 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     4381 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     4441 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     4501 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     4561 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     4621 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     4681 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4741 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     4801 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     4861 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     4921 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     4981 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     5041 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     5101 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     5161 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     5221 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     5281 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     5341 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     5401 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5461 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5521 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     5581 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     5641 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     5701 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     5761 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     5821 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     5881 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     5941 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     6001 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     6061 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     6121 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     6181 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
     6241 aatcgaagaa ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc
     6301 tgaagtcggt aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga
     6361 taaactggaa gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt
     6421 ctgggcacac gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc
     6481 ggacaaagcg ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg
     6541 caagctgatt gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct
     6601 gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc
     6661 gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat
     6721 tgctgctgac gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt
     6781 gggcgtggat aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa
     6841 caaacacatg aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga
     6901 aacagcgatg accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa
     6961 ttatggtgta acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt
     7021 gctgagcgca ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga
     7081 aaactatctg ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc
     7141 cgtagcgctg aagtcttacg aggaagagtt
//
