LOCUS Exported 6355 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses untagged human rhinovirus 3C protease in E. coli. ACCESSION . VERSION . KEYWORDS pSRK2706 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6355) AUTHORS Raran-Kurussi S, Waugh DS TITLE A dual protease approach for expression and affinity purification of recombinant proteins. JOURNAL Anal Biochem. 2016 Apr 19;504:30-37. doi: 10.1016/j.ab.2016.04.006. PUBMED 27105777 REFERENCE 2 (bases 1 to 6355) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6355 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 27..46 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 130..149 /label=T7 /note="T7 promoter, forward primer" promoter 130..148 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 149..173 /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)." protein_bind 214..238 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(812..836) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 856..897 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 907..924 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(981..999) /label=T7 Term /note="T7 terminator, reverse primer" terminator 995..1042 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(1217..1245) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(1276..1294) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1362..1466 /gene="bla" /label=AmpR promoter CDS 1467..2327 /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(1685..1704) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2498..3086 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2987..3006 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3240..3257 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3272..3411 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3337..3356) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3497..3519 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3513..3704) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5016..6098) /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 6060..6079 /label=LacI-R /note="LacI, reverse primer" promoter complement(6099..6176) /gene="lacI" /label=lacI promoter ORIGIN 1 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 61 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc 121 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 181 aaataatttt gtttaacttt aagaaggaat atcacaagtt tgtacaaaaa agcaggcttt 241 aagaaggaga tatacatatg ggaccaaaca cagaatttgc actatccctg ttaaggaaaa 301 acataatgac tataacaacc tcaaagggag agttcacagg gttaggcata catgatcgtg 361 tctgtgtgat acccacacac gcacagcctg gtgatgatgt actagtaaat ggtcagaaaa 421 ttagagttaa ggataagtac aaattagtag atccagagaa cattaatcta gagcttacag 481 tgttgacttt agatagaaat gaaaaattca gagatatcag gggatttata tcagaagatc 541 tagaaggtgt ggatgccact ttggtagtac attcaaataa ctttaccaac actatcttag 601 aagttggccc tgtaacaatg gcaggactta ttaatttgag tagcaccccc actaacagaa 661 tgattcgtta tgattatgca acaaaaactg ggcagtgtgg aggtgtgctg tgtgctactg 721 gtaagatctt tggtattcat gttggcggta atggaagaca aggattttca gctcaactta 781 aaaaacaata ttttgtagag aaacaataat aacccagctt tcttgtacaa agtggtgatc 841 aattcgaagc ttgaaggtaa gcctatccct aaccctctcc tcggtctcga ttctacgcgt 901 accggtcatc atcaccatca ccattgagtt tgatccggct gctaacaaag cccgaaagga 961 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa 1021 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg 1081 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg 1141 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac 1201 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agaattaatt 1261 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 1321 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 1381 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 1441 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 1501 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 1561 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 1621 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 1681 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc 1741 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 1801 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 1861 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 1921 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 1981 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat 2041 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 2101 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 2161 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 2221 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 2281 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 2341 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 2401 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 2461 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 2521 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 2581 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 2641 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 2701 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 2761 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 2821 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 2881 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 2941 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 3001 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 3061 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 3121 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 3181 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 3241 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 3301 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata 3361 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac 3421 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 3481 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 3541 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct 3601 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg 3661 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa 3721 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata 3781 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg 3841 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat 3901 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata 3961 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc 4021 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg 4081 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc 4141 aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc 4201 cgcgtgcggc tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc 4261 gcattcacag ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta 4321 gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca 4381 acgcggggag gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg 4441 tgctcgccga ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc 4501 tggtaagagc cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg 4561 acagcatggc ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg 4621 ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg 4681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga 4741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg 4801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta 4861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc 4921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct 4981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 5041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 5101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc 5161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga 5221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg 5281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt 5341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc 5401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct 5461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc 5521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga 5581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc 5641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg 5701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga 5761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg 5821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc 5881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt 5941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt 6001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg 6061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc 6121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg 6181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag 6241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc 6301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccg //