LOCUS Exported 7178 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4134414 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7178) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7178) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7178 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 602..622 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 623..649 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" polyA_signal 718..839 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(729..748) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 783..802 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 954..1000 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1030..1166 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1211..1282 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1508..2096) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1588..1607) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2596..3805 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3642..3661 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3809..4837 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA KE" polyA_signal 4864..4912 /note="synthetic polyadenylation signal" CDS 5178..6038 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5396..5415) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6051..6187 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6332..6403 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6408..6454 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6472..6520 /note="synthetic polyadenylation signal" enhancer 6601..6904 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6906..7108 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7059..7079 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7177..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatggca attgcctcag aatttagttc cctcccctcg tacgctgcgt tcgctaccgc 61 ccaagaggca tatgagcagg ccgtagccaa cggggacagc gaagtggttc ttaagaaact 121 gaagaaatct ttgaatgtgg caaagagcga gtttgatcgg gacgcggcaa tgcagaggaa 181 gctggaaaaa atggctgatc aggctatgac acagatgtac aaacaagccc gatccgagga 241 caagagagct aaagtcactt ctgccatgca aacgatgcta ttcaccatgt tacgtaagct 301 ggataacgac gcactcaata acataatcaa taacgcccgc gatggttgtg tgccattgaa 361 tatcattcct cttactacag cggccaagct gatggtcgtt atcccggact ataacacata 421 caaaaatacc tgcgacggca ccactttcac gtatgctagc gccctgtggg aaatacagca 481 ggtagtggat gccgattcaa agattgtgca gcttagtgag atctccatgg acaacagtcc 541 caacttggct tggcctttaa tcgtcacagc tctcagagcc aatagcgcag ttaaactgca 601 agaaaacctt tactttcagt cgtaccccta tgacgtacca gattatgcgt aaggatccgc 661 aggcctctgc tagcttgact gactgagata cagcgtacct tcagctcaca gacatgataa 721 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt 781 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta 841 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt 901 aaagcaagta aaacctctac aaatgtggta ttggcccatc tctatcggta tcgtagcata 961 accccttggg gcctctaaac gggtcttgag gggttttttg tgcccctcgg gccggattgc 1021 tatctaccgg cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc 1081 cacatatgcc agattcagca acggatacgg cttccccaac ttgcccactt ccatacgtgt 1141 cctccttacc agaaatttat ccttaaggtc gtcagctatc ctgcaggcga tctctcgatt 1201 tcgatcaaga cattccttta atggtctttt ctggacacca ctaggggtca gaagtagttc 1261 atcaaacttt cttccctccc taatctcatt ggttaccttg ggctatcgaa acttaattaa 1321 ccagtcaagt cagctacttg gcgagatcga cttgtctggg tttcgactac gctcagaatt 1381 gcgtcagtca agttcgatct ggtccttgct attgcacccg ttctccgatt acgagtttca 1441 tttaaatcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 1501 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 1561 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 1621 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 1681 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 1741 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 1801 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1861 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1921 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 1981 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 2041 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 2101 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 2161 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 2221 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 2281 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcatttaaat 2341 ttccgaactc tccaaggccc tcgtcggaaa atcttcaaac ctttcgtccg atccatcttg 2401 caggctacct ctcgaacgaa ctatcgcaag tctcttggcc ggccttgcgc cttggctatt 2461 gcttggcagc gcctatcgcc aggtattact ccaatcccga atatccgaga tcgggatcac 2521 ccgagagaag ttcaacctac atcctcaatc ccgatctatc cgagatccga ggaatatcga 2581 aatcggggcg cgcctggcct ccgcgccggg ttttggcgcc tcccgcgggc gcccccctcg 2641 tcacggcgag cgctgccacg tcagacgaag ggcgcaggag cgtcctgatc cttccgcccg 2701 gacgctcagg acagcggccc gctgctcata agactcggcc ttagaacccc agtatcagca 2761 gaaggacatt ttaggacggg acttgggtga ctctagggca ctggttttct ttccagagag 2821 cggaacaggc gaggaaaagt agtcccttct cggcgattct gcggagggat ctccgtgggg 2881 cggtgaacgc cgatgattat ataaggacgc gccgggtgtg gcacagctag ttccgtcgca 2941 gccgggattt gggtcgcggt tcttgtttgt ggatcgctgt gatcgtcact tggtgagtag 3001 cgggctgctg ggctggccgg ggctttcgtg gccgccgggc cgctcggtgg gacggaagcg 3061 tgtggagaga ccgccaaggg ctgtagtctg ggtccgcgag caaggttgcc ctgaactggg 3121 ggttgggggg agcgcagcaa aatggcggct gttcccgagt cttgaatgga agacgcttgt 3181 gaggcgggct gtgaggtcgt tgaaacaagg tggggggcat ggtgggcggc aagaacccaa 3241 ggtcttgagc ccttcgctaa tgcgggaaag ctcttattcg ggtgagatgg gctgggcacc 3301 atctggggac cctgacgtga agtttgtcac tgactggaga actcggtttg tcgtctgttg 3361 cgggggcggc agttatggcg gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg 3421 ccctcgtcgt gtcgtgacgt cacccgttct gttggcttat aatgcagggt ggggccacct 3481 gccggtaggt gtgcggtagg cttttctccg tcgcaggacg cagggttcgg gcctagggta 3541 ggctctcctg aatcgacagg cgccggacct ctggtgaggg gagggataag tgaggcgtca 3601 gtttctttgg tcggttttat gtacctatct tcttaagtag ctgaagctcc ggttttgaac 3661 tatgcgctcg gggttggcga gtgtgttttg tgaagttttt taggcacctt ttgaaatgta 3721 atcatttggg tcaatatgta attttcagtg ttagacttgt aaattgtccg ctaaattctg 3781 gccgtttttg gcttttttgt tagacaacat gggtaaaaag cctgaactca ccgcgacgtc 3841 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga 3901 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt 3961 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatccgc 4021 cgcgctcccg attccggaag tgcttgacat tggggagttc agcgagagcc tgacctattg 4081 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc 4141 tgttctgcag ccggtcgcgg aggcaatgga tgccatcgct gccgccgatc ttagccagac 4201 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt 4261 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt 4321 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga 4381 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg 4441 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc 4501 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga 4561 gcggaggcat ccggagcttg caggatcgcc ccggctccgg gcgtatatgc tccgcattgg 4621 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca 4681 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc 4741 ccgcagaagc gccgccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa 4801 ccgacgcccc agcactcgtc cgagggcaaa ggaataagct agtatgtaag cctagtctta 4861 gataataaaa tcgctatcca tcgaagatgg atgtgtgttg gttttttgtg tgtgtaacgc 4921 taggcgcgcc tggtgtaccg agaacgatcc tctcagtgcg agtctcgacg atccatatcg 4981 ttgcttggca gtcagccagt cggaatccag cttgggaccc aggaagtcca atcgtcagat 5041 attgtactca agcctggtca cggcagcgta ccgatctgtt taaacctaga tattgatagt 5101 ctgatcggtc aacgtataat cgagtcctag cttttgcaaa catctatcaa gagacaggat 5161 cagcaggagg ctttcgcatg agtattcaac atttccgtgt cgcccttatt cccttttttg 5221 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 5281 aagatcagtt gggtgcgcga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 5341 ttgagagttt tcgccccgaa gaacgctttc caatgatgag cacttttaaa gttctgctat 5401 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 5461 attctcagaa tgacttggtt gagtattcac cagtcacaga aaagcatctt acggatggca 5521 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 5581 tacttctgac aacgattgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 5641 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 5701 agcgtgacac cacgatgcct gtagcaatgg caacaacctt gcgtaaacta ttaactggcg 5761 aactacttac tctagcttcc cggcaacagt tgatagactg gatggaggcg gataaagttg 5821 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 5881 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 5941 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 6001 tcgctgagat aggtgcctca ctgattaagc attggtaacc gattctaggt gcattggcgc 6061 agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc cagattcagc 6121 aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac cagaaattta 6181 tccttaagat cgtttaaact cgactctggc tctatcgaat ctccgtcgtt tcgagcttac 6241 gcgaacagcc gtggcgctca tttgctcgtc gggcatcgaa tctcgtcagc tatcgtcagc 6301 ttaccttttt ggcagcgatc gcggctcccg acatcttgga ccattagctc cacaggtatc 6361 ttcttccctc tagtggtcat aacagcagct tcagctacct ctcaattcaa aaaacccctc 6421 aagacccgtt tagaggcccc aaggggttat gctatcaatc gttgcgttac acacacaaaa 6481 aaccaacaca catccatctt cgatggatag cgattttatt atctaactgc tgatcgagtg 6541 tagccagatc tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 6601 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 6661 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 6721 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 6781 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 6841 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 6901 catgctgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 6961 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 7021 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 7081 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatca gatctttgtc 7141 gatcctacca tccactcgac acacccgcca gcggccgc //