LOCUS Exported 6740 bp ds-DNA circular SYN 11-FEB-2022 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4134491 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6740) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6740) AUTHORS . TITLE Direct Submission JOURNAL Exported Feb 11, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6740 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..304 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 306..508 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 459..479 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 577..586 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1180..1200 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1201..1227 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" polyA_signal 1280..1401 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1291..1310) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1345..1364 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1516..1562 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1592..1728 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1773..1844 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2070..2658) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2150..2169) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3158..4367 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4204..4223 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4371..4970 /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(4371..4390) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4867..4887 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4997..5045 /note="synthetic polyadenylation signal" CDS 5311..6171 /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(5529..5548) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6184..6320 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6472..6543 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6548..6594 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6612..6660 /note="synthetic polyadenylation signal" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 301 catgctgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatca gatctttgtc 541 gatcctacca tccactcgac acacccgcca gcggccgcca ccatggcaat tgcctcagaa 601 tttagttccc tcccctcgta cgctgcgttc gctaccgccc aagaggcata tgagcaggcc 661 gtagccaacg gggacagcga agtggttctt aagaaactga agaaatcttt gaatgtggca 721 aagagcgagt ttgatcggga cgcggcaatg cagaggaagc tggaaaaaat ggctgatcag 781 gctatgacac agatgtacaa acaagcccga tccgaggaca agagagctaa agtcacttct 841 gccatgcaaa cgatgctatt caccatgtta cgtaagctgg ataacgacgc actcaataac 901 ataatcaata acgcccgcga tggttgtgtg ccattgaata tcattcctct tactacagcg 961 gccaagctga tggtcgttat cccggactat aacacataca aaaatacctg cgacggcacc 1021 actttcacgt atgctagcgc cctgtgggaa atacagcagg tagtggatgc cgattcaaag 1081 attgtgcagc ttagtgagat ctccatggac aacagtccca acttggcttg gcctttaatc 1141 gtcacagctc tcagagccaa tagcgcagtt aaactgcaag aaaaccttta ctttcagtcg 1201 tacccctatg acgtaccaga ttatgcgtaa gctagcttga ctgactgaga tacagcgtac 1261 cttcagctca cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg 1321 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt 1381 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag 1441 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tattggccca 1501 tctctatcgg tatcgtagca taaccccttg gggcctctaa acgggtcttg aggggttttt 1561 tgtgcccctc gggccggatt gctatctacc ggcattggcg cagaaaaaaa tgcctgatgc 1621 gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac ggcttcccca 1681 acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaagg tcgtcagcta 1741 tcctgcaggc gatctctcga tttcgatcaa gacattcctt taatggtctt ttctggacac 1801 cactaggggt cagaagtagt tcatcaaact ttcttccctc cctaatctca ttggttacct 1861 tgggctatcg aaacttaatt aaccagtcaa gtcagctact tggcgagatc gacttgtctg 1921 ggtttcgact acgctcagaa ttgcgtcagt caagttcgat ctggtccttg ctattgcacc 1981 cgttctccga ttacgagttt catttaaatc atgtgagcaa aaggccagca aaaggccagg 2041 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 2101 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 2161 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 2221 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 2281 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 2341 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 2401 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 2461 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 2521 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 2581 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 2641 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 2701 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 2761 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 2821 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 2881 tcatccatag ttgcatttaa atttccgaac tctccaaggc cctcgtcgga aaatcttcaa 2941 acctttcgtc cgatccatct tgcaggctac ctctcgaacg aactatcgca agtctcttgg 3001 ccggccttgc gccttggcta ttgcttggca gcgcctatcg ccaggtatta ctccaatccc 3061 gaatatccga gatcgggatc acccgagaga agttcaacct acatcctcaa tcccgatcta 3121 tccgagatcc gaggaatatc gaaatcgggg cgcgcctggc ctccgcgccg ggttttggcg 3181 cctcccgcgg gcgcccccct cgtcacggcg agcgctgcca cgtcagacga agggcgcagg 3241 agcgtcctga tccttccgcc cggacgctca ggacagcggc ccgctgctca taagactcgg 3301 ccttagaacc ccagtatcag cagaaggaca ttttaggacg ggacttgggt gactctaggg 3361 cactggtttt ctttccagag agcggaacag gcgaggaaaa gtagtccctt ctcggcgatt 3421 ctgcggaggg atctccgtgg ggcggtgaac gccgatgatt atataaggac gcgccgggtg 3481 tggcacagct agttccgtcg cagccgggat ttgggtcgcg gttcttgttt gtggatcgct 3541 gtgatcgtca cttggtgagt agcgggctgc tgggctggcc ggggctttcg tggccgccgg 3601 gccgctcggt gggacggaag cgtgtggaga gaccgccaag ggctgtagtc tgggtccgcg 3661 agcaaggttg ccctgaactg ggggttgggg ggagcgcagc aaaatggcgg ctgttcccga 3721 gtcttgaatg gaagacgctt gtgaggcggg ctgtgaggtc gttgaaacaa ggtggggggc 3781 atggtgggcg gcaagaaccc aaggtcttga gcccttcgct aatgcgggaa agctcttatt 3841 cgggtgagat gggctgggca ccatctgggg accctgacgt gaagtttgtc actgactgga 3901 gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg cggtgccgtt gggcagtgca 3961 cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac gtcacccgtt ctgttggctt 4021 ataatgcagg gtggggccac ctgccggtag gtgtgcggta ggcttttctc cgtcgcagga 4081 cgcagggttc gggcctaggg taggctctcc tgaatcgaca ggcgccggac ctctggtgag 4141 gggagggata agtgaggcgt cagtttcttt ggtcggtttt atgtacctat cttcttaagt 4201 agctgaagct ccggttttga actatgcgct cggggttggc gagtgtgttt tgtgaagttt 4261 tttaggcacc ttttgaaatg taatcatttg ggtcaatatg taattttcag tgttagactt 4321 gtaaattgtc cgctaaattc tggccgtttt tggctttttt gttagacaac atgaccgagt 4381 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg 4441 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg 4501 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg 4561 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag 4621 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg 4681 ccgcgcagca acagatggag ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt 4741 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg 4801 tgctccccgg agtggaggcc gccgagcgcg ccggggtgcc cgccttcctg gagacctccg 4861 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg 4921 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctag gctagtatgt 4981 aagcctagtc ttagataata aaatcgctat ccatcgaaga tggatgtgtg ttggtttttt 5041 gtgtgtgtaa cgctaggcgc gcctggtgta ccgagaacga tcctctcagt gcgagtctcg 5101 acgatccata tcgttgcttg gcagtcagcc agtcggaatc cagcttggga cccaggaagt 5161 ccaatcgtca gatattgtac tcaagcctgg tcacggcagc gtaccgatct gtttaaacct 5221 agatattgat agtctgatcg gtcaacgtat aatcgagtcc tagcttttgc aaacatctat 5281 caagagacag gatcagcagg aggctttcgc atgagtattc aacatttccg tgtcgccctt 5341 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 5401 gtaaaagatg ctgaagatca gttgggtgcg cgagtgggtt acatcgaact ggatctcaac 5461 agcggtaaga tccttgagag ttttcgcccc gaagaacgct ttccaatgat gagcactttt 5521 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 5581 cgccgcatac actattctca gaatgacttg gttgagtatt caccagtcac agaaaagcat 5641 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 5701 actgcggcca acttacttct gacaacgatt ggaggaccga aggagctaac cgcttttttg 5761 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 5821 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac cttgcgtaaa 5881 ctattaactg gcgaactact tactctagct tcccggcaac agttgataga ctggatggag 5941 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 6001 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 6061 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 6121 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta accgattcta 6181 ggtgcattgg cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata ctcccacata 6241 tgccagattc agcaacggat acggcttccc caacttgccc acttccatac gtgtcctcct 6301 taccagaaat ttatccttaa gatcccgaat cgtttaaact cgactctggc tctatcgaat 6361 ctccgtcgtt tcgagcttac gcgaacagcc gtggcgctca tttgctcgtc gggcatcgaa 6421 tctcgtcagc tatcgtcagc ttaccttttt ggcagcgatc gcggctcccg acatcttgga 6481 ccattagctc cacaggtatc ttcttccctc tagtggtcat aacagcagct tcagctacct 6541 ctcaattcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctatcaatc 6601 gttgcgttac acacacaaaa aaccaacaca catccatctt cgatggatag cgattttatt 6661 atctaactgc tgatcgagtg tagccagatc tagtaatcaa ttacggggtc attagttcat 6721 agcccatata tggagttccg //