LOCUS Exported 7223 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4133991 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7223) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7223) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7223 /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..694 /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" polyA_signal 763..884 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(774..793) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 828..847 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 999..1045 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1075..1211 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1256..1327 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1553..2141) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1633..1652) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2641..3850 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3687..3706 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3854..4882 /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 4909..4957 /note="synthetic polyadenylation signal" CDS 5223..6083 /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(5441..5460) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6096..6232 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6377..6448 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6453..6499 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6517..6565 /note="synthetic polyadenylation signal" enhancer 6646..6949 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6951..7153 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7104..7124 /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 cagtcgaaga ggcgatggaa aaagaatttt atcgcggtga gtgcagccaa 661 cagattcaaa aagatatcct cgtctggtgc cctctaagga tccgcaggcc tctgctagct 721 tgactgactg agatacagcg taccttcagc tcacagacat gataagatac attgatgagt 781 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg 841 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca 901 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc 961 tctacaaatg tggtattggc ccatctctat cggtatcgta gcataacccc ttggggcctc 1021 taaacgggtc ttgaggggtt ttttgtgccc ctcgggccgg attgctatct accggcattg 1081 gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat atgccagatt 1141 cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa 1201 tttatcctta aggtcgtcag ctatcctgca ggcgatctct cgatttcgat caagacattc 1261 ctttaatggt cttttctgga caccactagg ggtcagaagt agttcatcaa actttcttcc 1321 ctccctaatc tcattggtta ccttgggcta tcgaaactta attaaccagt caagtcagct 1381 acttggcgag atcgacttgt ctgggtttcg actacgctca gaattgcgtc agtcaagttc 1441 gatctggtcc ttgctattgc acccgttctc cgattacgag tttcatttaa atcatgtgag 1501 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 1561 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1621 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1681 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1741 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1801 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 1861 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 1921 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 1981 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 2041 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 2101 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 2161 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 2221 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 2281 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 2341 tctcagcgat ctgtctattt cgttcatcca tagttgcatt taaatttccg aactctccaa 2401 ggccctcgtc ggaaaatctt caaacctttc gtccgatcca tcttgcaggc tacctctcga 2461 acgaactatc gcaagtctct tggccggcct tgcgccttgg ctattgcttg gcagcgccta 2521 tcgccaggta ttactccaat cccgaatatc cgagatcggg atcacccgag agaagttcaa 2581 cctacatcct caatcccgat ctatccgaga tccgaggaat atcgaaatcg gggcgcgcct 2641 ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcgtcacg gcgagcgctg 2701 ccacgtcaga cgaagggcgc aggagcgtcc tgatccttcc gcccggacgc tcaggacagc 2761 ggcccgctgc tcataagact cggccttaga accccagtat cagcagaagg acattttagg 2821 acgggacttg ggtgactcta gggcactggt tttctttcca gagagcggaa caggcgagga 2881 aaagtagtcc cttctcggcg attctgcgga gggatctccg tggggcggtg aacgccgatg 2941 attatataag gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc 3001 gcggttcttg tttgtggatc gctgtgatcg tcacttggtg agtagcgggc tgctgggctg 3061 gccggggctt tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg agagaccgcc 3121 aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa ctgggggttg gggggagcgc 3181 agcaaaatgg cggctgttcc cgagtcttga atggaagacg cttgtgaggc gggctgtgag 3241 gtcgttgaaa caaggtgggg ggcatggtgg gcggcaagaa cccaaggtct tgagcccttc 3301 gctaatgcgg gaaagctctt attcgggtga gatgggctgg gcaccatctg gggaccctga 3361 cgtgaagttt gtcactgact ggagaactcg gtttgtcgtc tgttgcgggg gcggcagtta 3421 tggcggtgcc gttgggcagt gcacccgtac ctttgggagc gcgcgccctc gtcgtgtcgt 3481 gacgtcaccc gttctgttgg cttataatgc agggtggggc cacctgccgg taggtgtgcg 3541 gtaggctttt ctccgtcgca ggacgcaggg ttcgggccta gggtaggctc tcctgaatcg 3601 acaggcgccg gacctctggt gaggggaggg ataagtgagg cgtcagtttc tttggtcggt 3661 tttatgtacc tatcttctta agtagctgaa gctccggttt tgaactatgc gctcggggtt 3721 ggcgagtgtg ttttgtgaag ttttttaggc accttttgaa atgtaatcat ttgggtcaat 3781 atgtaatttt cagtgttaga cttgtaaatt gtccgctaaa ttctggccgt ttttggcttt 3841 tttgttagac aacatgggta aaaagcctga actcaccgcg acgtctgtcg agaagtttct 3901 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg 3961 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga 4021 tggtttctac aaagatcgtt atgtttatcg gcactttgca tccgccgcgc tcccgattcc 4081 ggaagtgctt gacattgggg agttcagcga gagcctgacc tattgcatct cccgccgtgc 4141 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt 4201 cgcggaggca atggatgcca tcgctgccgc cgatcttagc cagacgagcg ggttcggccc 4261 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc 4321 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc 4381 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt 4441 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat 4501 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg 4561 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga 4621 gcttgcagga tcgccccggc tccgggcgta tatgctccgc attggtcttg accaactcta 4681 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc 4741 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgccgc 4801 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac 4861 tcgtccgagg gcaaaggaat aagctagtat gtaagcctag tcttagataa taaaatcgct 4921 atccatcgaa gatggatgtg tgttggtttt ttgtgtgtgt aacgctaggc gcgcctggtg 4981 taccgagaac gatcctctca gtgcgagtct cgacgatcca tatcgttgct tggcagtcag 5041 ccagtcggaa tccagcttgg gacccaggaa gtccaatcgt cagatattgt actcaagcct 5101 ggtcacggca gcgtaccgat ctgtttaaac ctagatattg atagtctgat cggtcaacgt 5161 ataatcgagt cctagctttt gcaaacatct atcaagagac aggatcagca ggaggctttc 5221 gcatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 5281 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 5341 cgcgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 5401 ccgaagaacg ctttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 5461 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 5521 tggttgagta ttcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 5581 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 5641 ttggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 5701 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 5761 tgcctgtagc aatggcaaca accttgcgta aactattaac tggcgaacta cttactctag 5821 cttcccggca acagttgata gactggatgg aggcggataa agttgcagga ccacttctgc 5881 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 5941 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 6001 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 6061 cctcactgat taagcattgg taaccgattc taggtgcatt ggcgcagaaa aaaatgcctg 6121 atgcgacgct gcgcgtctta tactcccaca tatgccagat tcagcaacgg atacggcttc 6181 cccaacttgc ccacttccat acgtgtcctc cttaccagaa atttatcctt aagatcgttt 6241 aaactcgact ctggctctat cgaatctccg tcgtttcgag cttacgcgaa cagccgtggc 6301 gctcatttgc tcgtcgggca tcgaatctcg tcagctatcg tcagcttacc tttttggcag 6361 cgatcgcggc tcccgacatc ttggaccatt agctccacag gtatcttctt ccctctagtg 6421 gtcataacag cagcttcagc tacctctcaa ttcaaaaaac ccctcaagac ccgtttagag 6481 gccccaaggg gttatgctat caatcgttgc gttacacaca caaaaaacca acacacatcc 6541 atcttcgatg gatagcgatt ttattatcta actgctgatc gagtgtagcc agatctagta 6601 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac 6661 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac 6721 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt 6781 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat 6841 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga 6901 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc tgatgcggtt 6961 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca 7021 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg 7081 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 7141 tataagcaga gctggtttag tgaaccgtca gatcagatct ttgtcgatcc taccatccac 7201 tcgacacacc cgccagcggc cgc //