LOCUS Exported 6778 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp8. ACCESSION . VERSION . KEYWORDS pGBW-m4133437 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6778) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6778) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6778 /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 747..868 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(758..777) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 812..831 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 983..1029 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1059..1195 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1240..1311 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1537..2125) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1617..1636) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2625..3834 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3671..3690 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3838..4437 /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(3838..3857) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4334..4354 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4464..4512 /note="synthetic polyadenylation signal" CDS 4778..5638 /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(4996..5015) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5651..5787 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 5932..6003 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6008..6054 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6072..6120 /note="synthetic polyadenylation signal" enhancer 6201..6504 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6506..6708 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6659..6679 /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 cctctaagct agcttgactg actgagatac 721 agcgtacctt cagctcacag acatgataag atacattgat gagtttggac aaaccacaac 781 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt 841 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca 901 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat 961 tggcccatct ctatcggtat cgtagcataa ccccttgggg cctctaaacg ggtcttgagg 1021 ggttttttgt gcccctcggg ccggattgct atctaccggc attggcgcag aaaaaaatgc 1081 ctgatgcgac gctgcgcgtc ttatactccc acatatgcca gattcagcaa cggatacggc 1141 ttccccaact tgcccacttc catacgtgtc ctccttacca gaaatttatc cttaaggtcg 1201 tcagctatcc tgcaggcgat ctctcgattt cgatcaagac attcctttaa tggtcttttc 1261 tggacaccac taggggtcag aagtagttca tcaaactttc ttccctccct aatctcattg 1321 gttaccttgg gctatcgaaa cttaattaac cagtcaagtc agctacttgg cgagatcgac 1381 ttgtctgggt ttcgactacg ctcagaattg cgtcagtcaa gttcgatctg gtccttgcta 1441 ttgcacccgt tctccgatta cgagtttcat ttaaatcatg tgagcaaaag gccagcaaaa 1501 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 1561 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 1621 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 1681 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 1741 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 1801 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 1861 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 1921 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 1981 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 2041 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 2101 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 2161 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 2221 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 2281 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 2341 atttcgttca tccatagttg catttaaatt tccgaactct ccaaggccct cgtcggaaaa 2401 tcttcaaacc tttcgtccga tccatcttgc aggctacctc tcgaacgaac tatcgcaagt 2461 ctcttggccg gccttgcgcc ttggctattg cttggcagcg cctatcgcca ggtattactc 2521 caatcccgaa tatccgagat cgggatcacc cgagagaagt tcaacctaca tcctcaatcc 2581 cgatctatcc gagatccgag gaatatcgaa atcggggcgc gcctggcctc cgcgccgggt 2641 tttggcgcct cccgcgggcg cccccctcgt cacggcgagc gctgccacgt cagacgaagg 2701 gcgcaggagc gtcctgatcc ttccgcccgg acgctcagga cagcggcccg ctgctcataa 2761 gactcggcct tagaacccca gtatcagcag aaggacattt taggacggga cttgggtgac 2821 tctagggcac tggttttctt tccagagagc ggaacaggcg aggaaaagta gtcccttctc 2881 ggcgattctg cggagggatc tccgtggggc ggtgaacgcc gatgattata taaggacgcg 2941 ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg 3001 gatcgctgtg atcgtcactt ggtgagtagc gggctgctgg gctggccggg gctttcgtgg 3061 ccgccgggcc gctcggtggg acggaagcgt gtggagagac cgccaagggc tgtagtctgg 3121 gtccgcgagc aaggttgccc tgaactgggg gttgggggga gcgcagcaaa atggcggctg 3181 ttcccgagtc ttgaatggaa gacgcttgtg aggcgggctg tgaggtcgtt gaaacaaggt 3241 ggggggcatg gtgggcggca agaacccaag gtcttgagcc cttcgctaat gcgggaaagc 3301 tcttattcgg gtgagatggg ctgggcacca tctggggacc ctgacgtgaa gtttgtcact 3361 gactggagaa ctcggtttgt cgtctgttgc gggggcggca gttatggcgg tgccgttggg 3421 cagtgcaccc gtacctttgg gagcgcgcgc cctcgtcgtg tcgtgacgtc acccgttctg 3481 ttggcttata atgcagggtg gggccacctg ccggtaggtg tgcggtaggc ttttctccgt 3541 cgcaggacgc agggttcggg cctagggtag gctctcctga atcgacaggc gccggacctc 3601 tggtgagggg agggataagt gaggcgtcag tttctttggt cggttttatg tacctatctt 3661 cttaagtagc tgaagctccg gttttgaact atgcgctcgg ggttggcgag tgtgttttgt 3721 gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt 3781 tagacttgta aattgtccgc taaattctgg ccgtttttgg cttttttgtt agacaacatg 3841 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc 3901 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc 3961 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc 4021 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc 4081 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc 4141 cggctggccg cgcagcaaca gatggagggc ctcctggcgc cgcaccggcc caaggagccc 4201 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc 4261 gccgtcgtgc tccccggagt ggaggccgcc gagcgcgccg gggtgcccgc cttcctggag 4321 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac 4381 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctaggct 4441 agtatgtaag cctagtctta gataataaaa tcgctatcca tcgaagatgg atgtgtgttg 4501 gttttttgtg tgtgtaacgc taggcgcgcc tggtgtaccg agaacgatcc tctcagtgcg 4561 agtctcgacg atccatatcg ttgcttggca gtcagccagt cggaatccag cttgggaccc 4621 aggaagtcca atcgtcagat attgtactca agcctggtca cggcagcgta ccgatctgtt 4681 taaacctaga tattgatagt ctgatcggtc aacgtataat cgagtcctag cttttgcaaa 4741 catctatcaa gagacaggat cagcaggagg ctttcgcatg agtattcaac atttccgtgt 4801 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 4861 ggtgaaagta aaagatgctg aagatcagtt gggtgcgcga gtgggttaca tcgaactgga 4921 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgctttc caatgatgag 4981 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 5041 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtattcac cagtcacaga 5101 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 5161 tgataacact gcggccaact tacttctgac aacgattgga ggaccgaagg agctaaccgc 5221 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 5281 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacctt 5341 gcgtaaacta ttaactggcg aactacttac tctagcttcc cggcaacagt tgatagactg 5401 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 5461 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg 5521 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 5581 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaacc 5641 gattctaggt gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc 5701 ccacatatgc cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg 5761 tcctccttac cagaaattta tccttaagat cgtttaaact cgactctggc tctatcgaat 5821 ctccgtcgtt tcgagcttac gcgaacagcc gtggcgctca tttgctcgtc gggcatcgaa 5881 tctcgtcagc tatcgtcagc ttaccttttt ggcagcgatc gcggctcccg acatcttgga 5941 ccattagctc cacaggtatc ttcttccctc tagtggtcat aacagcagct tcagctacct 6001 ctcaattcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctatcaatc 6061 gttgcgttac acacacaaaa aaccaacaca catccatctt cgatggatag cgattttatt 6121 atctaactgc tgatcgagtg tagccagatc tagtaatcaa ttacggggtc attagttcat 6181 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg 6241 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata 6301 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 6361 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc 6421 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 6481 gtattagtca tcgctattac catgctgatg cggttttggc agtacatcaa tgggcgtgga 6541 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 6601 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 6661 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac 6721 cgtcagatca gatctttgtc gatcctacca tccactcgac acacccgcca gcggccgc //