LOCUS       sequence-372934-        6389 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 6389)
  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 6389)
  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..6389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(143..934,935..1003))
                     /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     766..785
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1004..1108)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1176..1194
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1221..1226,1227..1243,1244..1249)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(1424..1471)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1467..1485
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2261..2275)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     RBS             2294..2316
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2331..2355
                     /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     complement(2355..2374)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2356..2374)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2458..2477)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2683..2760
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2761..3843
                     /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(2780..2799)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5155..5346
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5340..5362)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5448..5587
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5503..5522
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5602..5619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5773..6361)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5853..5872)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
       61 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
      121 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
      181 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
      241 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
      301 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
      361 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
      421 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
      481 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
      541 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
      601 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
      661 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
      721 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac
      781 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
      841 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
      901 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
      961 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     1021 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     1081 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     1141 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     1201 gtcttcaaga attctcatgt ttgacagctt atcatcgata agctttaatg cggtagttta
     1261 tcacagttaa attgctaacg cagtcaggca ccgtgtatga aatctaacaa tgcgctcatc
     1321 gtcatcctcg gcaccgtcac cctggatgct gtaggcatag gcttggttat gccggtactg
     1381 ccgggcctct tgcgggatat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
     1441 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact
     1501 cagcttcctt tcgggctttg ttagcagccg gatcctcgag ctaatgatga tgatgatgta
     1561 gaaaactttg tatccactct gtgaaccttg ggacccgggc atacacccct gggcgattag
     1621 gcagtgcaca ttgatatcca aatgacgtca cgccagccag gagccatctg ttgttttctt
     1681 ggcacatgag tggtccgcct gaatccccct gacaagaatc tacccctcct gcatcatagc
     1741 ctgcacacac catattttcc gtaatgttat attctggcat ctgttgttga catttctcat
     1801 ttgatagaag gggaacgtca gcttcttgca gtacgtctgc agtagaacct tgatatataa
     1861 gtgcccccca gccagcaata gaacaaattc ttcctggggg aaaaacttga ttttcttctg
     1921 gtaagctaat aggctgtata taatctgtgt agttcacttt catttcaaga tgcatcatgg
     1981 caatgtcatt gttctttctc cgtttattgt agtgtcggtt tatgacaatt tggtcaatca
     2041 acctagtttc tatctgagga gaagtcagat ttgatgccat atgcaggcct agcactgctt
     2101 tccacttaga cggctccata tttctcccgt acacgcagtg ggcggccgac accagccaat
     2161 ccctgctcac cagagaagct ccgcagacct gttgatcgtc gaaatacaga gcaacgaccc
     2221 aaggccaggc tccttctctg gagtcacttc ctccgacaat cttgtcgtcg tcatctatag
     2281 ggcccatggt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
     2341 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
     2401 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
     2461 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
     2521 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
     2581 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
     2641 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
     2701 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     2761 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     2821 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     2881 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     2941 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     3001 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     3061 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     3121 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     3181 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     3241 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     3301 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     3361 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     3421 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     3481 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     3541 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     3601 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     3661 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     3721 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     3781 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     3841 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat
     3901 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     3961 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     4021 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     4081 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     4141 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt
     4201 cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc
     4261 cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca
     4321 tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcattggacc
     4381 gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat
     4441 tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg
     4501 ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga
     4561 attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac
     4621 atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg
     4681 tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg
     4741 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc
     4801 tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg
     4861 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca
     4921 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga
     4981 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa
     5041 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt
     5101 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc
     5161 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt
     5221 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac
     5281 gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     5341 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     5401 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     5461 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
     5521 tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     5581 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5641 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5701 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5761 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5821 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5881 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5941 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6001 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6061 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6121 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6181 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     6241 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6301 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6361 agaagatcct ttgatctttt ctacggggt
//
