LOCUS Exported 7323 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression plasmid for COVID-SARS2 NSP13 helicase. ACCESSION . VERSION . KEYWORDS COVID-SARS2 NSP13 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7323) TITLE Bacterial expression plasmid for Covid-SARS2 Nsp13 helicase JOURNAL Unpublished REFERENCE 2 (bases 1 to 7323) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7323 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1872..1889 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1942..1960) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1956..2003 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2040..2495 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2127..2146) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2337..2358 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2587..3402) /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 3310..3329 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 3394..3413 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 3524..4112 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4013..4032 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4266..4283 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4298..4437 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4363..4382) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4523..4545 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4539..4730) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5539..6621) /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 6583..6602 /label=LacI-R /note="LacI, reverse primer" promoter complement(6622..6699) /gene="lacI" /label=lacI promoter primer_bind 6905..6924 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 7008..7027 /label=T7 /note="T7 promoter, forward primer" promoter 7008..7026 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 7027..7051 /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 7066..7088 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 7099..7116 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 7126..7296 /codon_start=1 /product="Z domain of staphylococcal protein A, engineered to contain multiple positive charges (Hedhammar and Hober, 2007)" /label=Z-basic tag /note="useful for purifying tagged proteins by cation-exchange chromatography" /translation="VDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLL AEAKKLNDAQPK" CDS 7303..7323 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" ORIGIN 1 atggctgttg gtgcatgcgt tttgtgtaat agtcaaactt ctttgcgttg cggcgcctgc 61 atccgccgcc catttctgtg ctgtaaatgt tgctacgatc atgtgattag cacgtcgcat 121 aagctggtgc tgtcggtcaa cccgtatgtt tgtaatgcgc ccggttgtga cgtcacggat 181 gttacccagc tctatctggg tggtatgtct tactactgca agtcacataa gccgccgatt 241 tcatttcctc tctgcgcgaa tggccaggtg tttggtttgt ataaaaacac ctgcgtgggg 301 tctgataatg taaccgattt taacgcgatt gctacgtgtg actggaccaa cgccggcgat 361 tatatccttg ccaacacctg cactgaacgt cttaaactct tcgcggccga aaccctgaaa 421 gcaaccgaag aaacgttcaa actgagctat ggtatcgcga cagtacgcga agtactcagt 481 gaccgggagt tgcacctgtc gtgggaagtc ggtaaacctc gccccccgtt aaaccgcaac 541 tacgtgttca ccggctatcg cgttaccaaa aacagcaagg tgcaaattgg tgagtacacc 601 tttgagaaag gcgactatgg tgatgccgta gtataccgcg gcaccacgac ttacaaactg 661 aatgtgggtg attattttgt attaacctca cataccgtaa tgccgttgag cgcccccacc 721 ctcgtaccgc aggaacacta tgtacgtatc acgggtttat acccgacgct gaacatcagc 781 gatgagttta gcagtaacgt tgctaactac cagaaagtgg gtatgcaaaa atactcgacg 841 ttacagggcc cgccaggtac tggaaagtct catttcgcca tcggcttagc cttatactat 901 ccatcagctc gcattgttta cacggcctgc tctcatgctg cagtggacgc cttatgcgag 961 aaggcgttaa aatatctgcc gattgataaa tgctcccgta ttatcccggc gcgggcgcgg 1021 gtcgagtgtt ttgacaaatt taaagtgaac agcacactgg aacagtatgt tttttgtacg 1081 gtgaatgctc tcccagagac aactgccgat atcgtcgtgt tcgacgaaat cagcatggcc 1141 actaactacg atctgtcggt cgttaacgcg cgtctccgcg ctaagcatta tgtttacatt 1201 ggcgatccgg cccagctgcc ggctccacgc acgctcctga ctaaaggtac tctggaaccg 1261 gaatatttta atagcgtatg ccgtctgatg aaaacgattg gacctgatat gttcctggga 1321 acttgccgcc ggtgtccggc cgagatcgtt gatacggttt ctgctctggt ttatgacaat 1381 aaattaaaag ctcacaaaga taaaagcgcg caatgcttca aaatgtttta taaaggtgta 1441 attacccacg atgtgagtag cgcgattaac cgcccgcaaa ttggcgttgt ccgtgaattc 1501 ctgacccgga atcctgcgtg gcgcaaagca gtgttcatta gcccatataa tagtcaaaac 1561 gcggtagcgt ccaagatttt gggtctgccg acacaaaccg tggactccag ccagggcagt 1621 gagtacgact atgtgatttt tacccagacc actgagacag cacactcctg caacgtgaac 1681 cgcttcaatg tagcgatcac ccgtgcgaag gtaggtattt tgtgcatcat gtccgaccgt 1741 gatctgtatg acaaattgca gtttaccagc ctggaaatcc ctcgccgtaa cgtggcaacc 1801 cttcaataac agtaaaggtg gatacggatc cgaattcgag ctccgtcgac aagcttgcgg 1861 ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg 1921 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta 1981 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga 2041 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 2101 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 2161 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 2221 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 2281 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 2341 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata 2401 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 2461 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg 2521 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat 2581 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt 2641 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca 2701 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat 2761 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt 2821 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac 2881 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg 2941 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg 3001 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc 3061 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca 3121 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag 3181 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt 3241 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg 3301 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa 3361 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact 3421 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt 3481 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 3541 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 3601 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 3661 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 3721 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 3781 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 3841 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 3901 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 3961 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 4021 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 4081 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 4141 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 4201 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 4261 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta 4321 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc 4381 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc 4441 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 4501 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 4561 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag 4621 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg 4681 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc 4741 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc 4801 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa 4861 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc 4921 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg 4981 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg 5041 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt 5101 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg 5161 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg 5221 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg 5281 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg 5341 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag 5401 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact 5461 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt 5521 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 5581 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt 5641 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga 5701 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt 5761 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat 5821 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg 5881 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg 5941 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt 6001 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg 6061 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg 6121 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag 6181 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag 6241 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt 6301 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc 6361 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt 6421 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat 6481 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct 6541 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc 6601 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca 6661 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct 6721 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg 6781 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg 6841 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc 6901 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg 6961 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac 7021 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa 7081 gaaggagata tacatatgca ccatcatcat catcattctt ctggtgtgga taacaagttc 7141 aacaaggagc gtcgaagagc tcgccgtgaa attcgccatc tgccgaacct gaaccgcgaa 7201 cagcgtcgcg catttattcg cagcctgcgc gatgatccga gccagagcgc gaacctgctg 7261 gcggaagcga agaagctgaa cgatgcgcag ccgaagggta ccgagaacct gtacttccaa 7321 tcc //