LOCUS Exported 7187 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4133751 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7187) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7187) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7187 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 596..616 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 617..658 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" polyA_signal 727..848 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(738..757) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 792..811 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 963..1009 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1039..1175 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1220..1291 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1517..2105) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1597..1616) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2605..3814 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3651..3670 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3818..4846 /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 4873..4921 /note="synthetic polyadenylation signal" CDS 5187..6047 /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(5405..5424) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6060..6196 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6341..6412 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6417..6463 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6481..6529 /note="synthetic polyadenylation signal" enhancer 6610..6913 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6915..7117 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7068..7088 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgata gcctcagagt ttagttccct tccatcatat gcagcttttg ctactgctca 61 agaagcttat gagcaggctg ttgctaatgg tgattctgaa gttgttctta aaaagttgaa 121 gaagtctttg aatgtggcta aatctgaatt tgaccgtgat gcagccatgc aacgtaagtt 181 ggaaaagatg gctgatcaag ctatgaccca aatgtataaa caggctagat ctgaggacaa 241 gagggcaaaa gttactagtg ctatgcagac aatgcttttc actatgctta gaaagttgga 301 taatgatgca ctcaacaaca ttatcaacaa tgcaagagat ggttgtgttc ccttgaacat 361 aatacctctt acaacagcag ccaaactaat ggttgtcata ccagactata acacatataa 421 aaatacgtgt gatggtacaa catttactta tgcatcagca ttgtgggaaa tccaacaggt 481 tgtagatgca gatagtaaaa ttgttcaact tagtgaaatt agtatggaca attcacctaa 541 tttagcatgg cctcttattg taacagcttt aagggccaat tctgctgtca aattagaaaa 601 cctttacttt cagtcgggga aacctatacc caatccactt ctcggcttag attctaccta 661 aggatccgca ggcctctgct agcttgactg actgagatac agcgtacctt cagctcacag 721 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat 781 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata 841 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg 901 aggtttttta aagcaagtaa aacctctaca aatgtggtat tggcccatct ctatcggtat 961 cgtagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgt gcccctcggg 1021 ccggattgct atctaccggc attggcgcag aaaaaaatgc ctgatgcgac gctgcgcgtc 1081 ttatactccc acatatgcca gattcagcaa cggatacggc ttccccaact tgcccacttc 1141 catacgtgtc ctccttacca gaaatttatc cttaaggtcg tcagctatcc tgcaggcgat 1201 ctctcgattt cgatcaagac attcctttaa tggtcttttc tggacaccac taggggtcag 1261 aagtagttca tcaaactttc ttccctccct aatctcattg gttaccttgg gctatcgaaa 1321 cttaattaac cagtcaagtc agctacttgg cgagatcgac ttgtctgggt ttcgactacg 1381 ctcagaattg cgtcagtcaa gttcgatctg gtccttgcta ttgcacccgt tctccgatta 1441 cgagtttcat ttaaatcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 1501 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 1561 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 1621 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 1681 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 1741 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 1801 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 1861 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 1921 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 1981 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 2041 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 2101 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 2161 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 2221 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 2281 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 2341 catttaaatt tccgaactct ccaaggccct cgtcggaaaa tcttcaaacc tttcgtccga 2401 tccatcttgc aggctacctc tcgaacgaac tatcgcaagt ctcttggccg gccttgcgcc 2461 ttggctattg cttggcagcg cctatcgcca ggtattactc caatcccgaa tatccgagat 2521 cgggatcacc cgagagaagt tcaacctaca tcctcaatcc cgatctatcc gagatccgag 2581 gaatatcgaa atcggggcgc gcctggcctc cgcgccgggt tttggcgcct cccgcgggcg 2641 cccccctcgt cacggcgagc gctgccacgt cagacgaagg gcgcaggagc gtcctgatcc 2701 ttccgcccgg acgctcagga cagcggcccg ctgctcataa gactcggcct tagaacccca 2761 gtatcagcag aaggacattt taggacggga cttgggtgac tctagggcac tggttttctt 2821 tccagagagc ggaacaggcg aggaaaagta gtcccttctc ggcgattctg cggagggatc 2881 tccgtggggc ggtgaacgcc gatgattata taaggacgcg ccgggtgtgg cacagctagt 2941 tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt 3001 ggtgagtagc gggctgctgg gctggccggg gctttcgtgg ccgccgggcc gctcggtggg 3061 acggaagcgt gtggagagac cgccaagggc tgtagtctgg gtccgcgagc aaggttgccc 3121 tgaactgggg gttgggggga gcgcagcaaa atggcggctg ttcccgagtc ttgaatggaa 3181 gacgcttgtg aggcgggctg tgaggtcgtt gaaacaaggt ggggggcatg gtgggcggca 3241 agaacccaag gtcttgagcc cttcgctaat gcgggaaagc tcttattcgg gtgagatggg 3301 ctgggcacca tctggggacc ctgacgtgaa gtttgtcact gactggagaa ctcggtttgt 3361 cgtctgttgc gggggcggca gttatggcgg tgccgttggg cagtgcaccc gtacctttgg 3421 gagcgcgcgc cctcgtcgtg tcgtgacgtc acccgttctg ttggcttata atgcagggtg 3481 gggccacctg ccggtaggtg tgcggtaggc ttttctccgt cgcaggacgc agggttcggg 3541 cctagggtag gctctcctga atcgacaggc gccggacctc tggtgagggg agggataagt 3601 gaggcgtcag tttctttggt cggttttatg tacctatctt cttaagtagc tgaagctccg 3661 gttttgaact atgcgctcgg ggttggcgag tgtgttttgt gaagtttttt aggcaccttt 3721 tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt tagacttgta aattgtccgc 3781 taaattctgg ccgtttttgg cttttttgtt agacaacatg ggtaaaaagc ctgaactcac 3841 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca 3901 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt 3961 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt 4021 tgcatccgcc gcgctcccga ttccggaagt gcttgacatt ggggagttca gcgagagcct 4081 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga 4141 actgcccgct gttctgcagc cggtcgcgga ggcaatggat gccatcgctg ccgccgatct 4201 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg 4261 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga 4321 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga 4381 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga 4441 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata 4501 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg 4561 ctacttcgag cggaggcatc cggagcttgc aggatcgccc cggctccggg cgtatatgct 4621 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc 4681 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac 4741 acaaatcgcc cgcagaagcg ccgccgtctg gaccgatggc tgtgtagaag tactcgccga 4801 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataagcta gtatgtaagc 4861 ctagtcttag ataataaaat cgctatccat cgaagatgga tgtgtgttgg ttttttgtgt 4921 gtgtaacgct aggcgcgcct ggtgtaccga gaacgatcct ctcagtgcga gtctcgacga 4981 tccatatcgt tgcttggcag tcagccagtc ggaatccagc ttgggaccca ggaagtccaa 5041 tcgtcagata ttgtactcaa gcctggtcac ggcagcgtac cgatctgttt aaacctagat 5101 attgatagtc tgatcggtca acgtataatc gagtcctagc ttttgcaaac atctatcaag 5161 agacaggatc agcaggaggc tttcgcatga gtattcaaca tttccgtgtc gcccttattc 5221 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 5281 aagatgctga agatcagttg ggtgcgcgag tgggttacat cgaactggat ctcaacagcg 5341 gtaagatcct tgagagtttt cgccccgaag aacgctttcc aatgatgagc acttttaaag 5401 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 5461 gcatacacta ttctcagaat gacttggttg agtattcacc agtcacagaa aagcatctta 5521 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 5581 cggccaactt acttctgaca acgattggag gaccgaagga gctaaccgct tttttgcaca 5641 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 5701 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaaccttg cgtaaactat 5761 taactggcga actacttact ctagcttccc ggcaacagtt gatagactgg atggaggcgg 5821 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 5881 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 5941 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 6001 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaaccg attctaggtg 6061 cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc cacatatgcc 6121 agattcagca acggatacgg cttccccaac ttgcccactt ccatacgtgt cctccttacc 6181 agaaatttat ccttaagatc gtttaaactc gactctggct ctatcgaatc tccgtcgttt 6241 cgagcttacg cgaacagccg tggcgctcat ttgctcgtcg ggcatcgaat ctcgtcagct 6301 atcgtcagct tacctttttg gcagcgatcg cggctcccga catcttggac cattagctcc 6361 acaggtatct tcttccctct agtggtcata acagcagctt cagctacctc tcaattcaaa 6421 aaacccctca agacccgttt agaggcccca aggggttatg ctatcaatcg ttgcgttaca 6481 cacacaaaaa accaacacac atccatcttc gatggatagc gattttatta tctaactgct 6541 gatcgagtgt agccagatct agtaatcaat tacggggtca ttagttcata gcccatatat 6601 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 6661 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 6721 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 6781 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 6841 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 6901 cgctattacc atgctgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 6961 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 7021 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 7081 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatcag 7141 atctttgtcg atcctaccat ccactcgaca cacccgccag cggccgc //