LOCUS Exported 6433 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp7. ACCESSION . VERSION . KEYWORDS pGBW-m4133530 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6433) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6433) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6433 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 251..271 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 272..349 /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 402..523 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(413..432) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 467..486 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 638..684 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 714..850 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 895..966 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1192..1780) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1272..1291) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2280..3489 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3326..3345 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3493..4092 /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(3493..3512) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3989..4009 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4119..4167 /note="synthetic polyadenylation signal" CDS 4433..5293 /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(4651..4670) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5306..5442 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 5587..5658 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 5663..5709 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 5727..5775 /note="synthetic polyadenylation signal" enhancer 5856..6159 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6161..6363 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6314..6334 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgaaa atgtcagatg taaagtgcac atcagtagtc ttactctcag ttttgcaaca 61 actcagagta gaatcatcat ctaaattgtg ggctcaatgt gtccagttac acaatgacat 121 tctcttagct aaagatacta ctgaagcctt tgaaaaaatg gtttcactac tttctgtttt 181 gctttccatg cagggtgctg tagacataaa caagctttgt gaagaaatgc tggacaacag 241 ggcaacctta gaaaaccttt actttcagtc gaagaggcga tggaaaaaga attttatcgc 301 ggtgagtgca gccaacagat tcaaaaagat atcctcgtct ggtgccctct aagctagctt 361 gactgactga gatacagcgt accttcagct cacagacatg ataagataca ttgatgagtt 421 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc 481 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat 541 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct 601 ctacaaatgt ggtattggcc catctctatc ggtatcgtag cataacccct tggggcctct 661 aaacgggtct tgaggggttt tttgtgcccc tcgggccgga ttgctatcta ccggcattgg 721 cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata ctcccacata tgccagattc 781 agcaacggat acggcttccc caacttgccc acttccatac gtgtcctcct taccagaaat 841 ttatccttaa ggtcgtcagc tatcctgcag gcgatctctc gatttcgatc aagacattcc 901 tttaatggtc ttttctggac accactaggg gtcagaagta gttcatcaaa ctttcttccc 961 tccctaatct cattggttac cttgggctat cgaaacttaa ttaaccagtc aagtcagcta 1021 cttggcgaga tcgacttgtc tgggtttcga ctacgctcag aattgcgtca gtcaagttcg 1081 atctggtcct tgctattgca cccgttctcc gattacgagt ttcatttaaa tcatgtgagc 1141 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 1201 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 1261 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 1321 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 1381 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 1441 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 1501 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 1561 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 1621 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 1681 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 1741 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 1801 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 1861 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 1921 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 1981 ctcagcgatc tgtctatttc gttcatccat agttgcattt aaatttccga actctccaag 2041 gccctcgtcg gaaaatcttc aaacctttcg tccgatccat cttgcaggct acctctcgaa 2101 cgaactatcg caagtctctt ggccggcctt gcgccttggc tattgcttgg cagcgcctat 2161 cgccaggtat tactccaatc ccgaatatcc gagatcggga tcacccgaga gaagttcaac 2221 ctacatcctc aatcccgatc tatccgagat ccgaggaata tcgaaatcgg ggcgcgcctg 2281 gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc ctcgtcacgg cgagcgctgc 2341 cacgtcagac gaagggcgca ggagcgtcct gatccttccg cccggacgct caggacagcg 2401 gcccgctgct cataagactc ggccttagaa ccccagtatc agcagaagga cattttagga 2461 cgggacttgg gtgactctag ggcactggtt ttctttccag agagcggaac aggcgaggaa 2521 aagtagtccc ttctcggcga ttctgcggag ggatctccgt ggggcggtga acgccgatga 2581 ttatataagg acgcgccggg tgtggcacag ctagttccgt cgcagccggg atttgggtcg 2641 cggttcttgt ttgtggatcg ctgtgatcgt cacttggtga gtagcgggct gctgggctgg 2701 ccggggcttt cgtggccgcc gggccgctcg gtgggacgga agcgtgtgga gagaccgcca 2761 agggctgtag tctgggtccg cgagcaaggt tgccctgaac tgggggttgg ggggagcgca 2821 gcaaaatggc ggctgttccc gagtcttgaa tggaagacgc ttgtgaggcg ggctgtgagg 2881 tcgttgaaac aaggtggggg gcatggtggg cggcaagaac ccaaggtctt gagcccttcg 2941 ctaatgcggg aaagctctta ttcgggtgag atgggctggg caccatctgg ggaccctgac 3001 gtgaagtttg tcactgactg gagaactcgg tttgtcgtct gttgcggggg cggcagttat 3061 ggcggtgccg ttgggcagtg cacccgtacc tttgggagcg cgcgccctcg tcgtgtcgtg 3121 acgtcacccg ttctgttggc ttataatgca gggtggggcc acctgccggt aggtgtgcgg 3181 taggcttttc tccgtcgcag gacgcagggt tcgggcctag ggtaggctct cctgaatcga 3241 caggcgccgg acctctggtg aggggaggga taagtgaggc gtcagtttct ttggtcggtt 3301 ttatgtacct atcttcttaa gtagctgaag ctccggtttt gaactatgcg ctcggggttg 3361 gcgagtgtgt tttgtgaagt tttttaggca ccttttgaaa tgtaatcatt tgggtcaata 3421 tgtaattttc agtgttagac ttgtaaattg tccgctaaat tctggccgtt tttggctttt 3481 ttgttagaca acatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc 3541 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc 3601 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc 3661 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg 3721 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc 3781 gagttgagcg gttcccggct ggccgcgcag caacagatgg agggcctcct ggcgccgcac 3841 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc 3901 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg ccgccgagcg cgccggggtg 3961 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc 4021 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag 4081 cccggtgcct aggctagtat gtaagcctag tcttagataa taaaatcgct atccatcgaa 4141 gatggatgtg tgttggtttt ttgtgtgtgt aacgctaggc gcgcctggtg taccgagaac 4201 gatcctctca gtgcgagtct cgacgatcca tatcgttgct tggcagtcag ccagtcggaa 4261 tccagcttgg gacccaggaa gtccaatcgt cagatattgt actcaagcct ggtcacggca 4321 gcgtaccgat ctgtttaaac ctagatattg atagtctgat cggtcaacgt ataatcgagt 4381 cctagctttt gcaaacatct atcaagagac aggatcagca ggaggctttc gcatgagtat 4441 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 4501 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cgcgagtggg 4561 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 4621 ctttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 4681 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 4741 ttcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 4801 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga ttggaggacc 4861 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 4921 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 4981 aatggcaaca accttgcgta aactattaac tggcgaacta cttactctag cttcccggca 5041 acagttgata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 5101 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 5161 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 5221 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 5281 taagcattgg taaccgattc taggtgcatt ggcgcagaaa aaaatgcctg atgcgacgct 5341 gcgcgtctta tactcccaca tatgccagat tcagcaacgg atacggcttc cccaacttgc 5401 ccacttccat acgtgtcctc cttaccagaa atttatcctt aagatcgttt aaactcgact 5461 ctggctctat cgaatctccg tcgtttcgag cttacgcgaa cagccgtggc gctcatttgc 5521 tcgtcgggca tcgaatctcg tcagctatcg tcagcttacc tttttggcag cgatcgcggc 5581 tcccgacatc ttggaccatt agctccacag gtatcttctt ccctctagtg gtcataacag 5641 cagcttcagc tacctctcaa ttcaaaaaac ccctcaagac ccgtttagag gccccaaggg 5701 gttatgctat caatcgttgc gttacacaca caaaaaacca acacacatcc atcttcgatg 5761 gatagcgatt ttattatcta actgctgatc gagtgtagcc agatctagta atcaattacg 5821 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc 5881 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc 5941 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact 6001 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat 6061 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact 6121 tggcagtaca tctacgtatt agtcatcgct attaccatgc tgatgcggtt ttggcagtac 6181 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac 6241 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac 6301 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga 6361 gctggtttag tgaaccgtca gatcagatct ttgtcgatcc taccatccac tcgacacacc 6421 cgccagcggc cgc //