LOCUS Exported 5361 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4134437 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5361) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 5361) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5361 /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 656..733 /codon_start=1 /product="calmodulin-binding peptide" /label=CBP /note="derived from skeletal muscle myosin light chain kinase; binds calmodulin with nanomolar affinity in the presence of calcium" /translation="KRRWKKNFIAVSAANRFKKISSSGAL" CDS 761..781 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" polyA_signal 1230..1351 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1241..1260) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1295..1314 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1466..1512 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1542..1678 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1723..1794 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2020..2608) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2100..2119) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS 3354..4214 /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(3572..3591) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 4227..4363 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 4515..4586 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 4591..4637 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 4655..4703 /note="synthetic polyadenylation signal" enhancer 4784..5087 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5089..5291 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 5242..5262 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 5360..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 cgggacggcg aatcccgggg ctgataaatc cctggaaaag 661 gagatggaag aagaacttta ttgcagtgtc tgcggctaac cggttcaaaa agatcagttc 721 aagcggcgcc ctagattacg acattcctac gaccgcctcc gagaatctct atttccaggg 781 tgaatttggc ttggcacaac atgacgaggc cgtcgataac aagtttaata aagaacagca 841 gaacgctttc tatgagatcc ttcacttacc aaatctgaac gaagagcaac gcaacgcttt 901 cattcagagc ctgaaagacg atccctcgca gtctgcaaat ctcctggccg aagccaagaa 961 gctgaatgac gcacaggctc caaaagttga taacaagttt aacaaggagc agcagaatgc 1021 tttctacgag atattgcacc ttcctaatct gaatgaggaa caaagaaatg catttatcca 1081 gtccctaaaa gacgatccat cacagagtgc caacctgctt gcggaagcca aaaagctcaa 1141 tgatgcccag gccccgaaag tagacgcgaa ccatcaataa gctagcttga ctgactgaga 1201 tacagcgtac cttcagctca cagacatgat aagatacatt gatgagtttg gacaaaccac 1261 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 1321 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 1381 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg 1441 tattggccca tctctatcgg tatcgtagca taaccccttg gggcctctaa acgggtcttg 1501 aggggttttt tgtgcccctc gggccggatt gctatctacc ggcattggcg cagaaaaaaa 1561 tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac 1621 ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaagg 1681 tcgtcagcta tcctgcaggc gatctctcga tttcgatcaa gacattcctt taatggtctt 1741 ttctggacac cactaggggt cagaagtagt tcatcaaact ttcttccctc cctaatctca 1801 ttggttacct tgggctatcg aaacttaatt aaccagtcaa gtcagctact tggcgagatc 1861 gacttgtctg ggtttcgact acgctcagaa ttgcgtcagt caagttcgat ctggtccttg 1921 ctattgcacc cgttctccga ttacgagttt catttaaatc atgtgagcaa aaggccagca 1981 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 2041 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 2101 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 2161 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 2221 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 2281 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 2341 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 2401 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 2461 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 2521 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 2581 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 2641 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 2701 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 2761 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 2821 tctatttcgt tcatccatag ttgcatttaa atttccgaac tctccaaggc cctcgtcgga 2881 aaatcttcaa acctttcgtc cgatccatct tgcaggctac ctctcgaacg aactatcgca 2941 agtctcttgg ccggccttgc gccttggcta ttgcttggca gcgcctatcg ccaggtatta 3001 ctccaatccc gaatatccga gatcgggatc acccgagaga agttcaacct acatcctcaa 3061 tcccgatcta tccgagatcc gaggaatatc gaaatcgggg cgcgcctggt gtaccgagaa 3121 cgatcctctc agtgcgagtc tcgacgatcc atatcgttgc ttggcagtca gccagtcgga 3181 atccagcttg ggacccagga agtccaatcg tcagatattg tactcaagcc tggtcacggc 3241 agcgtaccga tctgtttaaa cctagatatt gatagtctga tcggtcaacg tataatcgag 3301 tcctagcttt tgcaaacatc tatcaagaga caggatcagc aggaggcttt cgcatgagta 3361 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 3421 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcgcgagtgg 3481 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 3541 gctttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 3601 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 3661 attcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 3721 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg attggaggac 3781 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 3841 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 3901 caatggcaac aaccttgcgt aaactattaa ctggcgaact acttactcta gcttcccggc 3961 aacagttgat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 4021 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 4081 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 4141 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 4201 ttaagcattg gtaaccgatt ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc 4261 tgcgcgtctt atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg 4321 cccacttcca tacgtgtcct ccttaccaga aatttatcct taagatcccg aatcgtttaa 4381 actcgactct ggctctatcg aatctccgtc gtttcgagct tacgcgaaca gccgtggcgc 4441 tcatttgctc gtcgggcatc gaatctcgtc agctatcgtc agcttacctt tttggcagcg 4501 atcgcggctc ccgacatctt ggaccattag ctccacaggt atcttcttcc ctctagtggt 4561 cataacagca gcttcagcta cctctcaatt caaaaaaccc ctcaagaccc gtttagaggc 4621 cccaaggggt tatgctatca atcgttgcgt tacacacaca aaaaaccaac acacatccat 4681 cttcgatgga tagcgatttt attatctaac tgctgatcga gtgtagccag atctagtaat 4741 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg 4801 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 4861 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 4921 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg 4981 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact 5041 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatgctg atgcggtttt 5101 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 5161 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 5221 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 5281 taagcagagc tggtttagtg aaccgtcaga tcagatcttt gtcgatccta ccatccactc 5341 gacacacccg ccagcggccg c //