LOCUS Exported 7189 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 endoRNAse. ACCESSION . VERSION . KEYWORDS pGBW-m4133162 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7189) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7189) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7189 /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 1061..1105 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" polyA_signal 1158..1279 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1169..1188) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1223..1242 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1394..1440 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1470..1606 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1651..1722 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1948..2536) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2028..2047) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3036..4245 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4082..4101 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4249..4848 /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(4249..4268) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4745..4765 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4875..4923 /note="synthetic polyadenylation signal" CDS 5189..6049 /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(5407..5426) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6062..6198 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6343..6414 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6419..6465 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6483..6531 /note="synthetic polyadenylation signal" enhancer 6612..6915 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6917..7119 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7070..7090 /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 aaagaaaccg cggcagctaa 1081 gttcgagagg cagcacatgg acagttaagc tagcttgact gactgagata cagcgtacct 1141 tcagctcaca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 1201 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 1261 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg 1321 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta ttggcccatc 1381 tctatcggta tcgtagcata accccttggg gcctctaaac gggtcttgag gggttttttg 1441 tgcccctcgg gccggattgc tatctaccgg cattggcgca gaaaaaaatg cctgatgcga 1501 cgctgcgcgt cttatactcc cacatatgcc agattcagca acggatacgg cttccccaac 1561 ttgcccactt ccatacgtgt cctccttacc agaaatttat ccttaaggtc gtcagctatc 1621 ctgcaggcga tctctcgatt tcgatcaaga cattccttta atggtctttt ctggacacca 1681 ctaggggtca gaagtagttc atcaaacttt cttccctccc taatctcatt ggttaccttg 1741 ggctatcgaa acttaattaa ccagtcaagt cagctacttg gcgagatcga cttgtctggg 1801 tttcgactac gctcagaatt gcgtcagtca agttcgatct ggtccttgct attgcacccg 1861 ttctccgatt acgagtttca tttaaatcat gtgagcaaaa ggccagcaaa aggccaggaa 1921 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 1981 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 2041 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 2101 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 2161 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 2221 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 2281 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 2341 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 2401 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 2461 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 2521 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 2581 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 2641 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 2701 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 2761 atccatagtt gcatttaaat ttccgaactc tccaaggccc tcgtcggaaa atcttcaaac 2821 ctttcgtccg atccatcttg caggctacct ctcgaacgaa ctatcgcaag tctcttggcc 2881 ggccttgcgc cttggctatt gcttggcagc gcctatcgcc aggtattact ccaatcccga 2941 atatccgaga tcgggatcac ccgagagaag ttcaacctac atcctcaatc ccgatctatc 3001 cgagatccga ggaatatcga aatcggggcg cgcctggcct ccgcgccggg ttttggcgcc 3061 tcccgcgggc gcccccctcg tcacggcgag cgctgccacg tcagacgaag ggcgcaggag 3121 cgtcctgatc cttccgcccg gacgctcagg acagcggccc gctgctcata agactcggcc 3181 ttagaacccc agtatcagca gaaggacatt ttaggacggg acttgggtga ctctagggca 3241 ctggttttct ttccagagag cggaacaggc gaggaaaagt agtcccttct cggcgattct 3301 gcggagggat ctccgtgggg cggtgaacgc cgatgattat ataaggacgc gccgggtgtg 3361 gcacagctag ttccgtcgca gccgggattt gggtcgcggt tcttgtttgt ggatcgctgt 3421 gatcgtcact tggtgagtag cgggctgctg ggctggccgg ggctttcgtg gccgccgggc 3481 cgctcggtgg gacggaagcg tgtggagaga ccgccaaggg ctgtagtctg ggtccgcgag 3541 caaggttgcc ctgaactggg ggttgggggg agcgcagcaa aatggcggct gttcccgagt 3601 cttgaatgga agacgcttgt gaggcgggct gtgaggtcgt tgaaacaagg tggggggcat 3661 ggtgggcggc aagaacccaa ggtcttgagc ccttcgctaa tgcgggaaag ctcttattcg 3721 ggtgagatgg gctgggcacc atctggggac cctgacgtga agtttgtcac tgactggaga 3781 actcggtttg tcgtctgttg cgggggcggc agttatggcg gtgccgttgg gcagtgcacc 3841 cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt cacccgttct gttggcttat 3901 aatgcagggt ggggccacct gccggtaggt gtgcggtagg cttttctccg tcgcaggacg 3961 cagggttcgg gcctagggta ggctctcctg aatcgacagg cgccggacct ctggtgaggg 4021 gagggataag tgaggcgtca gtttctttgg tcggttttat gtacctatct tcttaagtag 4081 ctgaagctcc ggttttgaac tatgcgctcg gggttggcga gtgtgttttg tgaagttttt 4141 taggcacctt ttgaaatgta atcatttggg tcaatatgta attttcagtg ttagacttgt 4201 aaattgtccg ctaaattctg gccgtttttg gcttttttgt tagacaacat gaccgagtac 4261 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc 4321 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag 4381 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg 4441 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg 4501 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc 4561 gcgcagcaac agatggaggg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc 4621 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg 4681 ctccccggag tggaggccgc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg 4741 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg 4801 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctaggc tagtatgtaa 4861 gcctagtctt agataataaa atcgctatcc atcgaagatg gatgtgtgtt ggttttttgt 4921 gtgtgtaacg ctaggcgcgc ctggtgtacc gagaacgatc ctctcagtgc gagtctcgac 4981 gatccatatc gttgcttggc agtcagccag tcggaatcca gcttgggacc caggaagtcc 5041 aatcgtcaga tattgtactc aagcctggtc acggcagcgt accgatctgt ttaaacctag 5101 atattgatag tctgatcggt caacgtataa tcgagtccta gcttttgcaa acatctatca 5161 agagacagga tcagcaggag gctttcgcat gagtattcaa catttccgtg tcgcccttat 5221 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 5281 aaaagatgct gaagatcagt tgggtgcgcg agtgggttac atcgaactgg atctcaacag 5341 cggtaagatc cttgagagtt ttcgccccga agaacgcttt ccaatgatga gcacttttaa 5401 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 5461 ccgcatacac tattctcaga atgacttggt tgagtattca ccagtcacag aaaagcatct 5521 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 5581 tgcggccaac ttacttctga caacgattgg aggaccgaag gagctaaccg cttttttgca 5641 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 5701 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacct tgcgtaaact 5761 attaactggc gaactactta ctctagcttc ccggcaacag ttgatagact ggatggaggc 5821 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 5881 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 5941 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 6001 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac cgattctagg 6061 tgcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact cccacatatg 6121 ccagattcag caacggatac ggcttcccca acttgcccac ttccatacgt gtcctcctta 6181 ccagaaattt atccttaaga tcgtttaaac tcgactctgg ctctatcgaa tctccgtcgt 6241 ttcgagctta cgcgaacagc cgtggcgctc atttgctcgt cgggcatcga atctcgtcag 6301 ctatcgtcag cttacctttt tggcagcgat cgcggctccc gacatcttgg accattagct 6361 ccacaggtat cttcttccct ctagtggtca taacagcagc ttcagctacc tctcaattca 6421 aaaaacccct caagacccgt ttagaggccc caaggggtta tgctatcaat cgttgcgtta 6481 cacacacaaa aaaccaacac acatccatct tcgatggata gcgattttat tatctaactg 6541 ctgatcgagt gtagccagat ctagtaatca attacggggt cattagttca tagcccatat 6601 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 6661 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 6721 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 6781 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 6841 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 6901 atcgctatta ccatgctgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 6961 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 7021 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 7081 ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc 7141 agatctttgt cgatcctacc atccactcga cacacccgcc agcggccgc //