LOCUS Exported 7042 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression of Sars-CoV2 Nsp15 protein with His-tag and GST-tag. ACCESSION . VERSION . KEYWORDS pETM33_Nsp15 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7042) TITLE Ivarsson lab SARS-CoV-2 plasmids REFERENCE 2 (bases 1 to 7042) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7042 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 43..631 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 532..551 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 785..802 /label=L4440 /note="L4440 vector, forward primer" misc_feature 817..956 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(882..901) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 1042..1064 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(1058..1249) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(2058..3140) /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 3102..3121 /label=LacI-R /note="LacI, reverse primer" promoter complement(3141..3218) /gene="lacI" /label=lacI promoter primer_bind 3424..3443 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 3527..3546 /label=T7 /note="T7 promoter, forward primer" promoter 3527..3545 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 3546..3570 /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 3585..3607 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 3620..3637 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 3656..4309 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK" primer_bind 4267..4289 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 4316..4339 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 5433..5450 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5503..5521) /label=T7 Term /note="T7 terminator, reverse primer" terminator 5517..5564 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 5601..6056 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5688..5707) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5898..5919 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6148..6963) /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 6871..6890 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 6955..6974 /label=pENTR-R /note="pENTR vectors, reverse primer" ORIGIN 1 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 61 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 121 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 181 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 241 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 301 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 361 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 421 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 481 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 541 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 601 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 661 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 721 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 781 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac 841 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc 901 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 961 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 1021 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 1081 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 1141 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 1201 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 1261 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 1321 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 1381 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 1441 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 1501 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 1561 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 1621 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 1681 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg 1741 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt 1801 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt 1861 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga 1921 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg 1981 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta 2041 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 2101 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt 2161 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag 2221 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg 2281 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata 2341 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga 2401 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt 2461 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg 2521 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg 2581 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc 2641 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga 2701 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc 2761 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta 2821 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg 2881 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg 2941 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg 3001 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg 3061 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg 3121 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat 3181 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt 3241 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc 3301 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc 3361 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc 3421 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc 3481 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact 3541 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag 3601 aaggagatat accatgaaac atcaccatca ccatcacaac actagtagca attccatgtc 3661 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga 3721 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg 3781 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga 3841 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt 3901 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat 3961 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt 4021 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata 4081 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt 4141 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg 4201 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc 4261 tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat cggatctgga 4321 agttctgttc caggggccca tggacggtct gcagagcctg gagaacgttg cgtttaacgt 4381 ggttaacaag ggccacttcg acggtcagca aggcgaagtg ccggttagca tcattaacaa 4441 caccgtgtac accaaggttg acggtgtgga tgttgagctg tttgaaaaca aaaccaccct 4501 gccggtgaac gttgcgttcg agctgtgggc gaagcgtaac atcaaaccgg tgccggaagt 4561 taagattctg aacaacctgg gtgtggacat cgcggcgaac accgttattt gggactataa 4621 acgtgatgcg ccggcgcaca tcagcaccat tggcgtttgc agcatgaccg acatcgcgaa 4681 gaaaccgacc gaaaccatct gcgcgccgct gaccgtgttc tttgacggtc gtgtggatgg 4741 ccaggttgac ctgtttcgta acgcgcgtaa cggtgtgctg atcaccgagg gtagcgttaa 4801 aggcctgcag ccgagcgtgg gtccgaaaca agcgagcctg aacggtgtta ccctgattgg 4861 cgaagcggtg aagacccagt tcaactacta taagaaagtt gacggcgtgg ttcagcaact 4921 gccggaaacc tactttaccc agagccgtaa cctgcaagag ttcaagccgc gtagccaaat 4981 ggagatcgat tttctggaac tggcgatgga cgagttcatt gaacgttaca aactggaggg 5041 ttatgcgttt gaacacatcg tttacggcga tttcagccac agccaactgg gtggcctgca 5101 cctgctgatt ggtctggcga agcgtttcaa agagagcccg tttgagctgg aagatttcat 5161 cccgatggac agcaccgtga agaactattt tattaccgat gcgcagaccg gcagcagcaa 5221 atgcgtgtgc agcgttatcg acctgctgct ggacgatttc gttgaaatca ttaaaagcca 5281 ggacctgagc gtggtgagca aggtggttaa agtgaccatc gattacaccg agattagctt 5341 tatgctgtgg tgcaaggacg gtcacgtgga aaccttctat ccgaaactgt aagaattcga 5401 gctccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg 5461 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag 5521 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta 5581 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 5641 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 5701 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 5761 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 5821 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 5881 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 5941 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 6001 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt 6061 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 6121 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg 6181 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga 6241 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat 6301 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc 6361 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat 6421 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc 6481 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt 6541 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc 6601 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg 6661 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg 6721 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc 6781 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg 6841 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc 6901 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct 6961 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa 7021 tcccttaacg tgagttttcg tt //