LOCUS Exported 7979 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 endoRNAse. ACCESSION . VERSION . KEYWORDS pGBW-m4134141 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7979) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7979) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7979 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1040..1060 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1409..1426 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 1519..1640 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1530..1549) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1584..1603 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1755..1801 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1831..1967 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2012..2083 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2309..2897) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2389..2408) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3397..4606 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4443..4462 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4610..5638 /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 5665..5713 /note="synthetic polyadenylation signal" CDS 5979..6839 /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(6197..6216) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6852..6988 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 7133..7204 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 7209..7255 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7273..7321 /note="synthetic polyadenylation signal" enhancer 7402..7705 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7707..7909 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7860..7880 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgtta gaaaatgtgg cttttaatgt tgtaaataag ggacactttg atggacaaca 61 gggtgaagta ccagtttcta tcattaataa cactgtttac acaaaagttg atggtgttga 121 tgtagaattg tttgaaaata aaacaacatt acctgttaat gtagcatttg agctttgggc 181 taagcgcaac attaaaccag taccagaggt gaaaatactc aataatttgg gtgtggacat 241 tgctgctaat actgtgatct gggactacaa aagagatgct ccagcacata tatctactat 301 tggtgtttgt tctatgactg acatagccaa gaaaccaact gaaacgattt gtgcaccact 361 cactgtcttt tttgatggta gagttgatgg tcaagtagac ttatttagaa atgcccgtaa 421 tggtgttctt attacagaag gtagtgttaa aggtttacaa ccatctgtag gtcccaaaca 481 agctagtctt aatggagtca cattaattgg agaagccgta aaaacacagt tcaattatta 541 taagaaagtt gatggtgttg tccaacaatt acctgaaact tactttactc agagtagaaa 601 tttacaagaa tttaaaccca ggagtcaaat ggaaattgat ttcttagaat tagctatgga 661 tgaattcatt gaacggtata aattagaagg ctatgccttc gaacatatcg tttatggaga 721 ttttagtcat agtcagttag gtggtttaca tctactgatt ggactagcta aacgttttaa 781 ggaatcacct tttgaattag aagattttat tcctatggac agtacagtta aaaactattt 841 cataacagat gcgcaaacag gttcatctaa gtgtgtgtgt tctgttattg atttattact 901 tgatgatttt gttgaaataa taaaatccca agatttatct gtagtttcta aggttgtcaa 961 agtgactatt gactatacag aaatttcatt tatgctttgg tgtaaagatg gccatgtaga 1021 aacattttac ccaaaattag aaaaccttta ctttcagtcg atgagtgaca aaatcattca 1081 tctaaccgat gactcctttg atacagacgt gttgaaggcc gatggtgcga tactggtaga 1141 tttctgggct gaatggtgtg gcccgtgcaa gatgatcgca cccattctgg acgagatcgc 1201 cgacgagtat caaggaaaat taactgtcgc taagcttaat atagatcaga acccagggac 1261 ggcacctaaa tacggcatca ggggaatacc taccctcctg ctctttaaga atggggaagt 1321 tgccgcgaca aaagtgggag ccctgtctaa gggccagcta aaagagttct tggacgctaa 1381 cctggcaggt tcaggctcgg ggcacatgca ccatcaccat caccatagca gcgggctcgt 1441 gccacgcggc taaggatccg caggcctctg ctagcttgac tgactgagat acagcgtacc 1501 ttcagctcac agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc 1561 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta 1621 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg 1681 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt attggcccat 1741 ctctatcggt atcgtagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt 1801 gtgcccctcg ggccggattg ctatctaccg gcattggcgc agaaaaaaat gcctgatgcg 1861 acgctgcgcg tcttatactc ccacatatgc cagattcagc aacggatacg gcttccccaa 1921 cttgcccact tccatacgtg tcctccttac cagaaattta tccttaaggt cgtcagctat 1981 cctgcaggcg atctctcgat ttcgatcaag acattccttt aatggtcttt tctggacacc 2041 actaggggtc agaagtagtt catcaaactt tcttccctcc ctaatctcat tggttacctt 2101 gggctatcga aacttaatta accagtcaag tcagctactt ggcgagatcg acttgtctgg 2161 gtttcgacta cgctcagaat tgcgtcagtc aagttcgatc tggtccttgc tattgcaccc 2221 gttctccgat tacgagtttc atttaaatca tgtgagcaaa aggccagcaa aaggccagga 2281 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 2341 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 2401 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 2461 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2521 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 2581 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 2641 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 2701 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 2761 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2821 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2881 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 2941 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 3001 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 3061 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 3121 catccatagt tgcatttaaa tttccgaact ctccaaggcc ctcgtcggaa aatcttcaaa 3181 cctttcgtcc gatccatctt gcaggctacc tctcgaacga actatcgcaa gtctcttggc 3241 cggccttgcg ccttggctat tgcttggcag cgcctatcgc caggtattac tccaatcccg 3301 aatatccgag atcgggatca cccgagagaa gttcaaccta catcctcaat cccgatctat 3361 ccgagatccg aggaatatcg aaatcggggc gcgcctggcc tccgcgccgg gttttggcgc 3421 ctcccgcggg cgcccccctc gtcacggcga gcgctgccac gtcagacgaa gggcgcagga 3481 gcgtcctgat ccttccgccc ggacgctcag gacagcggcc cgctgctcat aagactcggc 3541 cttagaaccc cagtatcagc agaaggacat tttaggacgg gacttgggtg actctagggc 3601 actggttttc tttccagaga gcggaacagg cgaggaaaag tagtcccttc tcggcgattc 3661 tgcggaggga tctccgtggg gcggtgaacg ccgatgatta tataaggacg cgccgggtgt 3721 ggcacagcta gttccgtcgc agccgggatt tgggtcgcgg ttcttgtttg tggatcgctg 3781 tgatcgtcac ttggtgagta gcgggctgct gggctggccg gggctttcgt ggccgccggg 3841 ccgctcggtg ggacggaagc gtgtggagag accgccaagg gctgtagtct gggtccgcga 3901 gcaaggttgc cctgaactgg gggttggggg gagcgcagca aaatggcggc tgttcccgag 3961 tcttgaatgg aagacgcttg tgaggcgggc tgtgaggtcg ttgaaacaag gtggggggca 4021 tggtgggcgg caagaaccca aggtcttgag cccttcgcta atgcgggaaa gctcttattc 4081 gggtgagatg ggctgggcac catctgggga ccctgacgtg aagtttgtca ctgactggag 4141 aactcggttt gtcgtctgtt gcgggggcgg cagttatggc ggtgccgttg ggcagtgcac 4201 ccgtaccttt gggagcgcgc gccctcgtcg tgtcgtgacg tcacccgttc tgttggctta 4261 taatgcaggg tggggccacc tgccggtagg tgtgcggtag gcttttctcc gtcgcaggac 4321 gcagggttcg ggcctagggt aggctctcct gaatcgacag gcgccggacc tctggtgagg 4381 ggagggataa gtgaggcgtc agtttctttg gtcggtttta tgtacctatc ttcttaagta 4441 gctgaagctc cggttttgaa ctatgcgctc ggggttggcg agtgtgtttt gtgaagtttt 4501 ttaggcacct tttgaaatgt aatcatttgg gtcaatatgt aattttcagt gttagacttg 4561 taaattgtcc gctaaattct ggccgttttt ggcttttttg ttagacaaca tgggtaaaaa 4621 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc 4681 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg 4741 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt 4801 ttatcggcac tttgcatccg ccgcgctccc gattccggaa gtgcttgaca ttggggagtt 4861 cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct 4921 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggcaatgg atgccatcgc 4981 tgccgccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca 5041 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca 5101 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct 5161 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa 5221 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg 5281 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga 5341 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cccggctccg 5401 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt 5461 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac 5521 tgtcgggcgt acacaaatcg cccgcagaag cgccgccgtc tggaccgatg gctgtgtaga 5581 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaataagc 5641 tagtatgtaa gcctagtctt agataataaa atcgctatcc atcgaagatg gatgtgtgtt 5701 ggttttttgt gtgtgtaacg ctaggcgcgc ctggtgtacc gagaacgatc ctctcagtgc 5761 gagtctcgac gatccatatc gttgcttggc agtcagccag tcggaatcca gcttgggacc 5821 caggaagtcc aatcgtcaga tattgtactc aagcctggtc acggcagcgt accgatctgt 5881 ttaaacctag atattgatag tctgatcggt caacgtataa tcgagtccta gcttttgcaa 5941 acatctatca agagacagga tcagcaggag gctttcgcat gagtattcaa catttccgtg 6001 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 6061 tggtgaaagt aaaagatgct gaagatcagt tgggtgcgcg agtgggttac atcgaactgg 6121 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgcttt ccaatgatga 6181 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 6241 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtattca ccagtcacag 6301 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 6361 gtgataacac tgcggccaac ttacttctga caacgattgg aggaccgaag gagctaaccg 6421 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 6481 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacct 6541 tgcgtaaact attaactggc gaactactta ctctagcttc ccggcaacag ttgatagact 6601 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 6661 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 6721 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 6781 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 6841 cgattctagg tgcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact 6901 cccacatatg ccagattcag caacggatac ggcttcccca acttgcccac ttccatacgt 6961 gtcctcctta ccagaaattt atccttaaga tcgtttaaac tcgactctgg ctctatcgaa 7021 tctccgtcgt ttcgagctta cgcgaacagc cgtggcgctc atttgctcgt cgggcatcga 7081 atctcgtcag ctatcgtcag cttacctttt tggcagcgat cgcggctccc gacatcttgg 7141 accattagct ccacaggtat cttcttccct ctagtggtca taacagcagc ttcagctacc 7201 tctcaattca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctatcaat 7261 cgttgcgtta cacacacaaa aaaccaacac acatccatct tcgatggata gcgattttat 7321 tatctaactg ctgatcgagt gtagccagat ctagtaatca attacggggt cattagttca 7381 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 7441 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 7501 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 7561 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 7621 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 7681 cgtattagtc atcgctatta ccatgctgat gcggttttgg cagtacatca atgggcgtgg 7741 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 7801 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 7861 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa 7921 ccgtcagatc agatctttgt cgatcctacc atccactcga cacacccgcc agcggccgc //