LOCUS Exported 6334 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression of Sars-CoV2 Nsp3a_Ub1 protein with His-tag and GST-tag. ACCESSION . VERSION . KEYWORDS pETM33_Nsp3a_Ub1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6334) TITLE Ivarsson lab SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 6334) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6334 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 1..589 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 490..509 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 743..760 /label=L4440 /note="L4440 vector, forward primer" misc_feature 775..914 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(840..859) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 1000..1022 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(1016..1207) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(2016..3098) /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 3060..3079 /label=LacI-R /note="LacI, reverse primer" promoter complement(3099..3176) /gene="lacI" /label=lacI promoter primer_bind 3382..3401 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 3485..3504 /label=T7 /note="T7 promoter, forward primer" promoter 3485..3503 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 3504..3528 /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)." RBS 3543..3565 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 3578..3595 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 3614..4267 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK" primer_bind 4225..4247 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 4274..4297 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 4683..4700 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4753..4771) /label=T7 Term /note="T7 terminator, reverse primer" terminator 4767..4814 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 4851..5306 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4938..4957) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5148..5169 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(5398..6213) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 6121..6140 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 6205..6224 /label=pENTR-R /note="pENTR vectors, reverse primer" ORIGIN 1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat 781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt 841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt 1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca 1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt 1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga 1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac 1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg 1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc 1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt 1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc 1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac 1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg 1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga 1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg 1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta 1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc 1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct 1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc 2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga 2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg 2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt 2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc 2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct 2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc 2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga 2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc 2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg 2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga 2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg 2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc 2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt 2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt 3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg 3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc 3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg 3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag 3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc 3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg 3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt 3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg 3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata 3541 attttgttta actttaagaa ggagatatac catgaaacat caccatcacc atcacaacac 3601 tagtagcaat tccatgtccc ctatactagg ttattggaaa attaagggcc ttgtgcaacc 3661 cactcgactt cttttggaat atcttgaaga aaaatatgaa gagcatttgt atgagcgcga 3721 tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc 3781 ttattatatt gatggtgatg ttaaattaac acagtctatg gccatcatac gttatatagc 3841 tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt gcagagattt caatgcttga 3901 aggagcggtt ttggatatta gatacggtgt ttcgagaatt gcatatagta aagactttga 3961 aactctcaaa gttgattttc ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg 4021 tttatgtcat aaaacatatt taaatggtga tcatgtaacc catcctgact tcatgttgta 4081 tgacgctctt gatgttgttt tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt 4141 agtttgtttt aaaaaacgta ttgaagctat cccacaaatt gataagtact tgaaatccag 4201 caagtatata gcatggcctt tgcagggctg gcaagccacg tttggtggtg gcgaccatcc 4261 tccaaaatcg gatctggaag ttctgttcca ggggcccatg gacggtgcgc cgaccaaggt 4321 gaccttcggc gacgataccg tgatcgaggt tcagggttac aaaagcgtga acatcacctt 4381 tgagctggac gaacgtattg ataaggttct gaacgagaaa tgcagcgcgt ataccgtgga 4441 actgggcacc gaggttaacg agttcgcgtg cgtggttgcg gacgcggtga tcaagaccct 4501 gcaaccggtt agcgagctgc tgaccccgct gggtattgac ctggatgaat ggagcatggc 4561 gacctactac ctgttcgacg agagcggcga atttaaactg gcgagccaca tgtactgcag 4621 cttttatccg ccggatgaat aagaattcga gctccgtcga caagcttgcg gccgcactcg 4681 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt 4741 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 4801 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg 4861 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 4921 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 4981 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 5041 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg 5101 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 5161 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt 5221 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 5281 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc 5341 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta 5401 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc 5461 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag 5521 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat 5581 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga 5641 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc 5701 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc 5761 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg 5821 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc 5881 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc 5941 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag 6001 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa 6061 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt 6121 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct 6181 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta 6241 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 6301 cgtcagaccc cgtagaaaag atcaaaggat cttc //