LOCUS Exported 5805 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 endoRNAse. ACCESSION . VERSION . KEYWORDS pGBW-m4133194 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5805) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 5805) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5805 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1046..1066 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1100..1177 /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 1205..1225 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" polyA_signal 1674..1795 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1685..1704) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1739..1758 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1910..1956 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1986..2122 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2167..2238 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2464..3052) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2544..2563) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS 3798..4658 /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(4016..4035) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 4671..4807 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 4959..5030 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 5035..5081 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 5099..5147 /note="synthetic polyadenylation signal" enhancer 5228..5531 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 5533..5735 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 5686..5706 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgtca ctggagaacg ttgctttcaa tgtagtgaac aaggggcact ttgacggaca 61 gcaaggcgaa gtgccggtca gtatcataaa taatacggtg tacaccaaag tcgatggtgt 121 tgacgtggag ttatttgaaa acaagacaac tttgcctgtc aatgtggcct tcgagctgtg 181 ggcaaaacgc aacattaagc cagtacccga agtgaaaatc ctcaacaatc ttggcgttga 241 tattgccgca aacactgtta tctgggatta taagcgagat gcgcctgccc atatatccac 301 cattggagtg tgttctatga cagacatcgc taaaaagccc accgagacaa tttgcgcgcc 361 actcacggta ttctttgacg ggagggtcga cggtcaggtg gatctgttta gaaatgcccg 421 taacggcgta cttattactg aaggaagcgt gaaaggccta cagcccagtg ttgggccgaa 481 gcaagcaagc ctaaatggag tcacactgat cggagaggct gtgaaaaccc agttcaacta 541 ctataagaag gtcgacggcg tggttcagca gctgcctgag acatacttta cccaatcgcg 601 gaatctgcaa gaatttaagc ccagatccca gatggagatt gacttcttgg aactggccat 661 ggatgagttc atcgaacggt ataagctcga gggctacgcc ttcgagcaca tagtttatgg 721 ggatttttct cattcacagt taggcggtct gcacttgctc atcggattag cgaaacgctt 781 taaagagagc ccattcgaac ttgaagactt tatcccaatg gactcaactg tgaagaatta 841 cttcataact gacgctcaga cagggtccag caagtgcgtc tgttctgtta ttgatttgct 901 acttgatgat ttcgtggaga ttattaaatc acaggacctg tccgtggttt cgaaggtcgt 961 gaaagtaacc atcgactata cggaaattag tttcatgctt tggtgtaagg atggccatgt 1021 ggagacattt tatcctaagc tccaggaaaa cctttacttt cagtcgggac ggcgaatccc 1081 ggggctgata aatccctgga aaaggagatg gaagaagaac tttattgcag tgtctgcggc 1141 taaccggttc aaaaagatca gttcaagcgg cgccctagat tacgacattc ctacgaccgc 1201 ctccgagaat ctctatttcc agggtgaatt tggcttggca caacatgacg aggccgtcga 1261 taacaagttt aataaagaac agcagaacgc tttctatgag atccttcact taccaaatct 1321 gaacgaagag caacgcaacg ctttcattca gagcctgaaa gacgatccct cgcagtctgc 1381 aaatctcctg gccgaagcca agaagctgaa tgacgcacag gctccaaaag ttgataacaa 1441 gtttaacaag gagcagcaga atgctttcta cgagatattg caccttccta atctgaatga 1501 ggaacaaaga aatgcattta tccagtccct aaaagacgat ccatcacaga gtgccaacct 1561 gcttgcggaa gccaaaaagc tcaatgatgc ccaggccccg aaagtagacg cgaaccatca 1621 ataagctagc ttgactgact gagatacagc gtaccttcag ctcacagaca tgataagata 1681 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 1741 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 1801 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag 1861 caagtaaaac ctctacaaat gtggtattgg cccatctcta tcggtatcgt agcataaccc 1921 cttggggcct ctaaacgggt cttgaggggt tttttgtgcc cctcgggccg gattgctatc 1981 taccggcatt ggcgcagaaa aaaatgcctg atgcgacgct gcgcgtctta tactcccaca 2041 tatgccagat tcagcaacgg atacggcttc cccaacttgc ccacttccat acgtgtcctc 2101 cttaccagaa atttatcctt aaggtcgtca gctatcctgc aggcgatctc tcgatttcga 2161 tcaagacatt cctttaatgg tcttttctgg acaccactag gggtcagaag tagttcatca 2221 aactttcttc cctccctaat ctcattggtt accttgggct atcgaaactt aattaaccag 2281 tcaagtcagc tacttggcga gatcgacttg tctgggtttc gactacgctc agaattgcgt 2341 cagtcaagtt cgatctggtc cttgctattg cacccgttct ccgattacga gtttcattta 2401 aatcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 2461 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 2521 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 2581 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 2641 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 2701 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 2761 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 2821 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 2881 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 2941 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 3001 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 3061 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 3121 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 3181 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 3241 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcat ttaaatttcc 3301 gaactctcca aggccctcgt cggaaaatct tcaaaccttt cgtccgatcc atcttgcagg 3361 ctacctctcg aacgaactat cgcaagtctc ttggccggcc ttgcgccttg gctattgctt 3421 ggcagcgcct atcgccaggt attactccaa tcccgaatat ccgagatcgg gatcacccga 3481 gagaagttca acctacatcc tcaatcccga tctatccgag atccgaggaa tatcgaaatc 3541 ggggcgcgcc tggtgtaccg agaacgatcc tctcagtgcg agtctcgacg atccatatcg 3601 ttgcttggca gtcagccagt cggaatccag cttgggaccc aggaagtcca atcgtcagat 3661 attgtactca agcctggtca cggcagcgta ccgatctgtt taaacctaga tattgatagt 3721 ctgatcggtc aacgtataat cgagtcctag cttttgcaaa catctatcaa gagacaggat 3781 cagcaggagg ctttcgcatg agtattcaac atttccgtgt cgcccttatt cccttttttg 3841 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 3901 aagatcagtt gggtgcgcga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 3961 ttgagagttt tcgccccgaa gaacgctttc caatgatgag cacttttaaa gttctgctat 4021 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 4081 attctcagaa tgacttggtt gagtattcac cagtcacaga aaagcatctt acggatggca 4141 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 4201 tacttctgac aacgattgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 4261 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 4321 agcgtgacac cacgatgcct gtagcaatgg caacaacctt gcgtaaacta ttaactggcg 4381 aactacttac tctagcttcc cggcaacagt tgatagactg gatggaggcg gataaagttg 4441 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 4501 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 4561 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 4621 tcgctgagat aggtgcctca ctgattaagc attggtaacc gattctaggt gcattggcgc 4681 agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc cagattcagc 4741 aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac cagaaattta 4801 tccttaagat cccgaatcgt ttaaactcga ctctggctct atcgaatctc cgtcgtttcg 4861 agcttacgcg aacagccgtg gcgctcattt gctcgtcggg catcgaatct cgtcagctat 4921 cgtcagctta cctttttggc agcgatcgcg gctcccgaca tcttggacca ttagctccac 4981 aggtatcttc ttccctctag tggtcataac agcagcttca gctacctctc aattcaaaaa 5041 acccctcaag acccgtttag aggccccaag gggttatgct atcaatcgtt gcgttacaca 5101 cacaaaaaac caacacacat ccatcttcga tggatagcga ttttattatc taactgctga 5161 tcgagtgtag ccagatctag taatcaatta cggggtcatt agttcatagc ccatatatgg 5221 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 5281 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 5341 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 5401 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 5461 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 5521 ctattaccat gctgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 5581 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 5641 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 5701 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatcagat 5761 ctttgtcgat cctaccatcc actcgacaca cccgccagcg gccgc //