LOCUS Exported 6389 bp ds-DNA circular SYN 23-SEP-2022 DEFINITION Expresses the pentahistidine-tagged catalytic domain of bovine enteropeptidase in E. coli. ACCESSION . VERSION . KEYWORDS pET-15b-EK_C122S_His5 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 Sep 23, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6389 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(143..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 1221..1249 /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" terminator 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 //