LOCUS Exported 5020 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Allows cloning of gene of interest into bacterial expression vector with PreScission Protease cleavable N-terminal GST tag, and a retained N-terminal 10x His tag.. ACCESSION . VERSION . KEYWORDS pGEX6P1-N-His SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5020) TITLE Bacterial expression plasmids for production of recombinant proteins with a PreScission Protease cleavable GST tag and additional small affinity tags JOURNAL Unpublished REFERENCE 2 (bases 1 to 5020) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5020 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 183..211 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 219..235 /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)." CDS 258..911 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK" primer_bind 869..891 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 918..941 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 951..980 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" primer_bind complement(1070..1092) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(1237..1255) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1323..1427 /gene="bla" /label=AmpR promoter CDS 1428..2288 /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(1646..1665) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2459..3047 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2948..2967 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3201..3218 /label=L4440 /note="L4440 vector, forward primer" promoter 3291..3368 /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." CDS 3369..4451 /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 YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind complement(3388..3407) /label=LacI-R /note="LacI, reverse primer" protein_bind 4464..4485 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4500..4530 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4538..4554 /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 4562..4578 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4562..4578 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 4574..81 /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR QPSEAVVWLCRS" primer_bind complement(4594..4611) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4594..4610) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4603..4625) /label=M13/pUC Forward /note="In lacZ gene" ORIGIN 1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg 61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt 121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc 181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca 241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc 301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc 361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc 421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata 481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc 541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact 601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag 661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt 721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa 781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat 841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc 901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc catcatcatc 961 atcatcatca tcatcatcac cgatccccgg aattcccggg tcgactcgag cggccgcatc 1021 gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 1081 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 1141 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 1201 gcgatagcgg agtgtataat tcttgaagac gaaagggcct cgtgatacgc ctatttttat 1261 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 1321 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 1381 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 1441 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 1501 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 1561 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 1621 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg 1681 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 1741 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 1801 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 1861 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 1921 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg 1981 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 2041 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 2101 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 2161 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 2221 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 2281 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 2341 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 2401 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 2461 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 2521 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 2581 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 2641 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 2701 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 2761 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 2821 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 2881 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 2941 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 3001 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 3061 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 3121 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 3181 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc 3241 ggtattttct ccttacgcat ctgtgcggta tttcacaccg cataaattcc gacaccatcg 3301 aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg 3361 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc 3421 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag 3481 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg 3541 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc 3601 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga 3661 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac 3721 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag 3781 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca 3841 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg 3901 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc 3961 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag 4021 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg 4081 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta 4141 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag 4201 acagctcatg ttatatcccg ccgtcaacca ccatcaaaca ggattttcgc ctgctggggc 4261 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc 4321 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct 4381 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa 4441 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct 4501 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 4561 acaggaaaca gctatgacca tgattacgga ttcactggcc gtcgttttac aacgtcgtga 4621 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 4681 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 4741 tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa gctggctgga 4801 gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga tgcacggtta 4861 cgatgcgccc atctacacca acgtaaccta tcccattacg gtcaatccgc cgtttgttcc 4921 cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa gctggctaca 4981 ggaaggccag acgcgaatta tttttgatgg cgttggaatt //