LOCUS Exported 7090 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4133957 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7090) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7090) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7090 /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 965..982 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 1059..1180 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1070..1089) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1124..1143 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1295..1341 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1371..1507 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1552..1623 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1849..2437) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1929..1948) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2937..4146 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3983..4002 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4150..4749 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(4150..4169) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4646..4666 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4776..4824 /note="synthetic polyadenylation signal" CDS 5090..5950 /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(5308..5327) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5963..6099 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6244..6315 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6320..6366 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6384..6432 /note="synthetic polyadenylation signal" enhancer 6513..6816 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6818..7020 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6971..6991 /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 cagtcgatga gtgacaaaat cattcatcta accgatgact cctttgatac 661 agacgtgttg aaggccgatg gtgcgatact ggtagatttc tgggctgaat ggtgtggccc 721 gtgcaagatg atcgcaccca ttctggacga gatcgccgac gagtatcaag gaaaattaac 781 tgtcgctaag cttaatatag atcagaaccc agggacggca cctaaatacg gcatcagggg 841 aatacctacc ctcctgctct ttaagaatgg ggaagttgcc gcgacaaaag tgggagccct 901 gtctaagggc cagctaaaag agttcttgga cgctaacctg gcaggttcag gctcggggca 961 catgcaccat caccatcacc atagcagcgg gctcgtgcca cgcggctaag ctagcttgac 1021 tgactgagat acagcgtacc ttcagctcac agacatgata agatacattg atgagtttgg 1081 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat 1141 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca 1201 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta 1261 caaatgtggt attggcccat ctctatcggt atcgtagcat aaccccttgg ggcctctaaa 1321 cgggtcttga ggggtttttt gtgcccctcg ggccggattg ctatctaccg gcattggcgc 1381 agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc cagattcagc 1441 aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac cagaaattta 1501 tccttaaggt cgtcagctat cctgcaggcg atctctcgat ttcgatcaag acattccttt 1561 aatggtcttt tctggacacc actaggggtc agaagtagtt catcaaactt tcttccctcc 1621 ctaatctcat tggttacctt gggctatcga aacttaatta accagtcaag tcagctactt 1681 ggcgagatcg acttgtctgg gtttcgacta cgctcagaat tgcgtcagtc aagttcgatc 1741 tggtccttgc tattgcaccc gttctccgat tacgagtttc atttaaatca tgtgagcaaa 1801 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 1861 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 1921 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 1981 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 2041 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 2101 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 2161 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 2221 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 2281 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 2341 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 2401 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 2461 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 2521 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 2581 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 2641 agcgatctgt ctatttcgtt catccatagt tgcatttaaa tttccgaact ctccaaggcc 2701 ctcgtcggaa aatcttcaaa cctttcgtcc gatccatctt gcaggctacc tctcgaacga 2761 actatcgcaa gtctcttggc cggccttgcg ccttggctat tgcttggcag cgcctatcgc 2821 caggtattac tccaatcccg aatatccgag atcgggatca cccgagagaa gttcaaccta 2881 catcctcaat cccgatctat ccgagatccg aggaatatcg aaatcggggc gcgcctggcc 2941 tccgcgccgg gttttggcgc ctcccgcggg cgcccccctc gtcacggcga gcgctgccac 3001 gtcagacgaa gggcgcagga gcgtcctgat ccttccgccc ggacgctcag gacagcggcc 3061 cgctgctcat aagactcggc cttagaaccc cagtatcagc agaaggacat tttaggacgg 3121 gacttgggtg actctagggc actggttttc tttccagaga gcggaacagg cgaggaaaag 3181 tagtcccttc tcggcgattc tgcggaggga tctccgtggg gcggtgaacg ccgatgatta 3241 tataaggacg cgccgggtgt ggcacagcta gttccgtcgc agccgggatt tgggtcgcgg 3301 ttcttgtttg tggatcgctg tgatcgtcac ttggtgagta gcgggctgct gggctggccg 3361 gggctttcgt ggccgccggg ccgctcggtg ggacggaagc gtgtggagag accgccaagg 3421 gctgtagtct gggtccgcga gcaaggttgc cctgaactgg gggttggggg gagcgcagca 3481 aaatggcggc tgttcccgag tcttgaatgg aagacgcttg tgaggcgggc tgtgaggtcg 3541 ttgaaacaag gtggggggca tggtgggcgg caagaaccca aggtcttgag cccttcgcta 3601 atgcgggaaa gctcttattc gggtgagatg ggctgggcac catctgggga ccctgacgtg 3661 aagtttgtca ctgactggag aactcggttt gtcgtctgtt gcgggggcgg cagttatggc 3721 ggtgccgttg ggcagtgcac ccgtaccttt gggagcgcgc gccctcgtcg tgtcgtgacg 3781 tcacccgttc tgttggctta taatgcaggg tggggccacc tgccggtagg tgtgcggtag 3841 gcttttctcc gtcgcaggac gcagggttcg ggcctagggt aggctctcct gaatcgacag 3901 gcgccggacc tctggtgagg ggagggataa gtgaggcgtc agtttctttg gtcggtttta 3961 tgtacctatc ttcttaagta gctgaagctc cggttttgaa ctatgcgctc ggggttggcg 4021 agtgtgtttt gtgaagtttt ttaggcacct tttgaaatgt aatcatttgg gtcaatatgt 4081 aattttcagt gttagacttg taaattgtcc gctaaattct ggccgttttt ggcttttttg 4141 ttagacaaca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc 4201 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc 4261 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc 4321 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc 4381 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag 4441 ttgagcggtt cccggctggc cgcgcagcaa cagatggagg gcctcctggc gccgcaccgg 4501 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag 4561 ggtctgggca gcgccgtcgt gctccccgga gtggaggccg ccgagcgcgc cggggtgccc 4621 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc 4681 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc 4741 ggtgcctagg ctagtatgta agcctagtct tagataataa aatcgctatc catcgaagat 4801 ggatgtgtgt tggttttttg tgtgtgtaac gctaggcgcg cctggtgtac cgagaacgat 4861 cctctcagtg cgagtctcga cgatccatat cgttgcttgg cagtcagcca gtcggaatcc 4921 agcttgggac ccaggaagtc caatcgtcag atattgtact caagcctggt cacggcagcg 4981 taccgatctg tttaaaccta gatattgata gtctgatcgg tcaacgtata atcgagtcct 5041 agcttttgca aacatctatc aagagacagg atcagcagga ggctttcgca tgagtattca 5101 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 5161 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcgc gagtgggtta 5221 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgctt 5281 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 5341 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtattc 5401 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 5461 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgattg gaggaccgaa 5521 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 5581 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 5641 ggcaacaacc ttgcgtaaac tattaactgg cgaactactt actctagctt cccggcaaca 5701 gttgatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 5761 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 5821 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 5881 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 5941 gcattggtaa ccgattctag gtgcattggc gcagaaaaaa atgcctgatg cgacgctgcg 6001 cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc aacttgccca 6061 cttccatacg tgtcctcctt accagaaatt tatccttaag atcgtttaaa ctcgactctg 6121 gctctatcga atctccgtcg tttcgagctt acgcgaacag ccgtggcgct catttgctcg 6181 tcgggcatcg aatctcgtca gctatcgtca gcttaccttt ttggcagcga tcgcggctcc 6241 cgacatcttg gaccattagc tccacaggta tcttcttccc tctagtggtc ataacagcag 6301 cttcagctac ctctcaattc aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt 6361 atgctatcaa tcgttgcgtt acacacacaa aaaaccaaca cacatccatc ttcgatggat 6421 agcgatttta ttatctaact gctgatcgag tgtagccaga tctagtaatc aattacgggg 6481 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 6541 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 6601 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 6661 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 6721 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 6781 cagtacatct acgtattagt catcgctatt accatgctga tgcggttttg gcagtacatc 6841 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 6901 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 6961 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 7021 ggtttagtga accgtcagat cagatctttg tcgatcctac catccactcg acacacccgc 7081 cagcggccgc //