LOCUS       sequence-72040-e        6392 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the pentahistidine-tagged catalytic domain of bovine 
            enteropeptidase in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6392)
  AUTHORS   Skala W, Goettig P, Brandstetter H
  TITLE     Do-it-yourself histidine-tagged bovine enterokinase: A handy member 
            of the protein engineer's toolbox.
  JOURNAL   J Biotechnol. 2013 Dec;168(4):421-5. doi: 
            10.1016/j.jbiotec.2013.10.022. Epub 2013 Oct 30.
  PUBMED    24184090
REFERENCE   2  (bases 1 to 6392)
  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"; doi: 
            "10.1016/j.jbiotec.2013.10.022"; journalName: "J Biotechnol"; date: 
            "2013-12"; volume: "168"; issue: "4"; pages: "421-5"
COMMENT     Sequence Label: pET-15b-EK_C122S_His5
FEATURES             Location/Qualifiers
     source          1..6392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             53..67
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     primer_bind     complement(843..861)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      857..904
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1079..1084,1085..1101,1102..1107))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1134..1152)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1220..1324
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1325..1393,1394..2185)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1543..1562)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2356..2944
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2845..2864
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3098..3115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3130..3272
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3198..3217)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3358..3380
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3374..3565)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4877..5959)
                     /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     5921..5940
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5960..6037)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6243..6262
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6346..6365
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6346..6364
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6365..6389
                     /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)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggcccta tagatgacga
       61 cgacaagatt gtcggaggaa gtgactccag agaaggagcc tggccttggg tcgttgctct
      121 gtatttcgac gatcaacagg tctgcggagc ttctctggtg agcagggatt ggctggtgtc
      181 ggccgcccac tgcgtgtacg ggagaaatat ggagccgtct aagtggaaag cagtgctagg
      241 cctgcatatg gcatcaaatc tgacttctcc tcagatagaa actaggttga ttgaccaaat
      301 tgtcataaac cgacactaca ataaacggag aaagaacaat gacattgcca tgatgcatct
      361 tgaaatgaaa gtgaactaca cagattatat acagcctatt agcttaccag aagaaaatca
      421 agtttttccc ccaggaagaa tttgttctat tgctggctgg ggggcactta tatatcaagg
      481 ttctactgca gacgtactgc aagaagctga cgttcccctt ctatcaaatg agaaatgtca
      541 acaacagatg ccagaatata acattacgga aaatatggtg tgtgcaggct atgatgcagg
      601 aggggtagat tcttgtcagg gggattcagg cggaccactc atgtgccaag aaaacaacag
      661 atggctcctg gctggcgtga cgtcatttgg atatcaatgt gcactgccta atcgcccagg
      721 ggtgtatgcc cgggtcccaa ggttcacaga gtggatacaa agttttctac atcatcatca
      781 tcattagctc gaggatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
      841 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
      901 tttgctgaaa ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata
      961 accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg
     1021 ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat
     1081 taaagcttat cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt
     1141 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     1201 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1261 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     1321 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1381 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1441 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1501 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1561 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1621 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1681 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1741 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     1801 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     1861 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     1921 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     1981 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2041 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2101 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2161 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2221 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2281 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2341 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2401 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2461 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2521 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2581 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2641 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2701 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2761 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2821 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2881 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2941 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3001 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3061 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3121 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3181 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3241 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3301 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3361 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3421 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     3481 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     3541 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     3601 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     3661 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     3721 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     3781 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     3841 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     3901 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     3961 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4021 tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga
     4081 tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca
     4141 agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc
     4201 cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag
     4261 gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata
     4321 aatcgccgtg acgatcagcg gtccagtgat cgaagttagg ctggtaagag ccgcgagcga
     4381 tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc
     4441 gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg
     4501 cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc
     4561 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     4621 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     4681 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     4741 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     4801 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     4861 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4921 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     4981 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5041 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5101 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     5161 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5221 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5281 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5341 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5401 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5461 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5521 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5581 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     5641 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     5701 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5761 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     5821 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     5881 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     5941 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     6001 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     6061 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     6121 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     6181 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6241 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     6301 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
     6361 taggggaatt gtgagcggat aacaattccc ct
//
