LOCUS Exported 7700 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4134284 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7700) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7700) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7700 /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 650..727 /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 755..775 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" polyA_signal 1240..1361 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1251..1270) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1305..1324 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1476..1522 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1552..1688 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1733..1804 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2030..2618) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2110..2129) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3118..4327 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4164..4183 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4331..5359 /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 5386..5434 /note="synthetic polyadenylation signal" CDS 5700..6560 /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(5918..5937) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6573..6709 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6854..6925 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6930..6976 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6994..7042 /note="synthetic polyadenylation signal" enhancer 7123..7426 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7428..7630 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7581..7601 /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 cagtcgggac ggcgaatccc ggggctgata aatccctgga aaaggagatg 661 gaagaagaac tttattgcag tgtctgcggc taaccggttc aaaaagatca gttcaagcgg 721 cgccctagat tacgacattc ctacgaccgc ctccgagaat ctctatttcc agggtgaatt 781 tggcttggca caacatgacg aggccgtcga taacaagttt aataaagaac agcagaacgc 841 tttctatgag atccttcact taccaaatct gaacgaagag caacgcaacg ctttcattca 901 gagcctgaaa gacgatccct cgcagtctgc aaatctcctg gccgaagcca agaagctgaa 961 tgacgcacag gctccaaaag ttgataacaa gtttaacaag gagcagcaga atgctttcta 1021 cgagatattg caccttccta atctgaatga ggaacaaaga aatgcattta tccagtccct 1081 aaaagacgat ccatcacaga gtgccaacct gcttgcggaa gccaaaaagc tcaatgatgc 1141 ccaggccccg aaagtagacg cgaaccatca ataaggatcc gcaggcctct gctagcttga 1201 ctgactgaga tacagcgtac cttcagctca cagacatgat aagatacatt gatgagtttg 1261 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 1321 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc 1381 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct 1441 acaaatgtgg tattggccca tctctatcgg tatcgtagca taaccccttg gggcctctaa 1501 acgggtcttg aggggttttt tgtgcccctc gggccggatt gctatctacc ggcattggcg 1561 cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag 1621 caacggatac ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt 1681 atccttaagg tcgtcagcta tcctgcaggc gatctctcga tttcgatcaa gacattcctt 1741 taatggtctt ttctggacac cactaggggt cagaagtagt tcatcaaact ttcttccctc 1801 cctaatctca ttggttacct tgggctatcg aaacttaatt aaccagtcaa gtcagctact 1861 tggcgagatc gacttgtctg ggtttcgact acgctcagaa ttgcgtcagt caagttcgat 1921 ctggtccttg ctattgcacc cgttctccga ttacgagttt catttaaatc atgtgagcaa 1981 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 2041 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 2101 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 2161 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 2221 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 2281 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 2341 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 2401 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 2461 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 2521 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 2581 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 2641 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 2701 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 2761 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 2821 cagcgatctg tctatttcgt tcatccatag ttgcatttaa atttccgaac tctccaaggc 2881 cctcgtcgga aaatcttcaa acctttcgtc cgatccatct tgcaggctac ctctcgaacg 2941 aactatcgca agtctcttgg ccggccttgc gccttggcta ttgcttggca gcgcctatcg 3001 ccaggtatta ctccaatccc gaatatccga gatcgggatc acccgagaga agttcaacct 3061 acatcctcaa tcccgatcta tccgagatcc gaggaatatc gaaatcgggg cgcgcctggc 3121 ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct cgtcacggcg agcgctgcca 3181 cgtcagacga agggcgcagg agcgtcctga tccttccgcc cggacgctca ggacagcggc 3241 ccgctgctca taagactcgg ccttagaacc ccagtatcag cagaaggaca ttttaggacg 3301 ggacttgggt gactctaggg cactggtttt ctttccagag agcggaacag gcgaggaaaa 3361 gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac gccgatgatt 3421 atataaggac gcgccgggtg tggcacagct agttccgtcg cagccgggat ttgggtcgcg 3481 gttcttgttt gtggatcgct gtgatcgtca cttggtgagt agcgggctgc tgggctggcc 3541 ggggctttcg tggccgccgg gccgctcggt gggacggaag cgtgtggaga gaccgccaag 3601 ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg ggggttgggg ggagcgcagc 3661 aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg ctgtgaggtc 3721 gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc aaggtcttga gcccttcgct 3781 aatgcgggaa agctcttatt cgggtgagat gggctgggca ccatctgggg accctgacgt 3841 gaagtttgtc actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg 3901 cggtgccgtt gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac 3961 gtcacccgtt ctgttggctt ataatgcagg gtggggccac ctgccggtag gtgtgcggta 4021 ggcttttctc cgtcgcagga cgcagggttc gggcctaggg taggctctcc tgaatcgaca 4081 ggcgccggac ctctggtgag gggagggata agtgaggcgt cagtttcttt ggtcggtttt 4141 atgtacctat cttcttaagt agctgaagct ccggttttga actatgcgct cggggttggc 4201 gagtgtgttt tgtgaagttt tttaggcacc ttttgaaatg taatcatttg ggtcaatatg 4261 taattttcag tgttagactt gtaaattgtc cgctaaattc tggccgtttt tggctttttt 4321 gttagacaac atgggtaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 4381 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 4441 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4501 tttctacaaa gatcgttatg tttatcggca ctttgcatcc gccgcgctcc cgattccgga 4561 agtgcttgac attggggagt tcagcgagag cctgacctat tgcatctccc gccgtgcaca 4621 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 4681 ggaggcaatg gatgccatcg ctgccgccga tcttagccag acgagcgggt tcggcccatt 4741 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 4801 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 4861 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 4921 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 4981 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 5041 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 5101 tgcaggatcg ccccggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 5161 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 5221 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgccgccgt 5281 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 5341 tccgagggca aaggaataag ctagtatgta agcctagtct tagataataa aatcgctatc 5401 catcgaagat ggatgtgtgt tggttttttg tgtgtgtaac gctaggcgcg cctggtgtac 5461 cgagaacgat cctctcagtg cgagtctcga cgatccatat cgttgcttgg cagtcagcca 5521 gtcggaatcc agcttgggac ccaggaagtc caatcgtcag atattgtact caagcctggt 5581 cacggcagcg taccgatctg tttaaaccta gatattgata gtctgatcgg tcaacgtata 5641 atcgagtcct agcttttgca aacatctatc aagagacagg atcagcagga ggctttcgca 5701 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 5761 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcgc 5821 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 5881 aagaacgctt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 5941 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 6001 ttgagtattc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 6061 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgattg 6121 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 6181 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 6241 ctgtagcaat ggcaacaacc ttgcgtaaac tattaactgg cgaactactt actctagctt 6301 cccggcaaca gttgatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 6361 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 6421 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 6481 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 6541 cactgattaa gcattggtaa ccgattctag gtgcattggc gcagaaaaaa atgcctgatg 6601 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc 6661 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag atcgtttaaa 6721 ctcgactctg gctctatcga atctccgtcg tttcgagctt acgcgaacag ccgtggcgct 6781 catttgctcg tcgggcatcg aatctcgtca gctatcgtca gcttaccttt ttggcagcga 6841 tcgcggctcc cgacatcttg gaccattagc tccacaggta tcttcttccc tctagtggtc 6901 ataacagcag cttcagctac ctctcaattc aaaaaacccc tcaagacccg tttagaggcc 6961 ccaaggggtt atgctatcaa tcgttgcgtt acacacacaa aaaaccaaca cacatccatc 7021 ttcgatggat agcgatttta ttatctaact gctgatcgag tgtagccaga tctagtaatc 7081 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 7141 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 7201 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 7261 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 7321 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 7381 tcctacttgg cagtacatct acgtattagt catcgctatt accatgctga tgcggttttg 7441 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 7501 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 7561 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 7621 aagcagagct ggtttagtga accgtcagat cagatctttg tcgatcctac catccactcg 7681 acacacccgc cagcggccgc //