LOCUS Exported 8984 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 RNA-dependent RNA polymerase. ACCESSION . VERSION . KEYWORDS pGBW-m4134165 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8984) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 8984) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8984 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 2..204 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 155..175 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 3078..3098 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 3102..3167 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 3220..3341 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3231..3250) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3285..3304 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 3456..3502 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 3532..3668 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 3713..3784 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(4010..4598) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4090..4109) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 5098..6307 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 6144..6163 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 6311..6910 /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(6311..6330) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6807..6827 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6937..6985 /note="synthetic polyadenylation signal" CDS 7251..8111 /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(7469..7488) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 8124..8260 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 8412..8483 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 8488..8534 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 8552..8600 /note="synthetic polyadenylation signal" enhancer 8681..8984 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 ctgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatcagatc tttgtcgatc 241 ctaccatcca ctcgacacac ccgccagcgg ccgccaccat gtctgccgac gcacagtcgt 301 tcctgaatag agtttgtggt gtatccgcgg cccggctaac tccatgcgga accgggacga 361 gcacagatgt ggtgtaccga gcttttgata tctataacga caaagtcgct ggcttcgcca 421 agtttcttaa gaccaactgt tgcaggttcc aagagaaaga cgaagatgat aatctcatag 481 actcatactt tgtggttaag cgtcacactt ttagtaatta tcagcatgag gaaacaattt 541 ataacttgct gaaggactgc cctgccgtag caaaacacga cttctttaaa ttccgcatcg 601 atggcgatat ggtcccgcat attagcagac agcggttaac caagtacact atggcagacc 661 tggtgtacgc cctgcgacac ttcgacgagg gaaactgtga cacgttaaaa gaaatcctcg 721 tgacttataa ttgttgcgat gacgattatt tcaacaagaa agattggtac gattttgtcg 781 agaatcccga tatactcaga gtgtacgcta acctggggga aagggttcgc caggcactat 841 tgaagacagt ccaattttgc gacgcgatgc ggaatgccgg cattgtgggt gtcctgacac 901 ttgataacca ggacctgaat ggcaactggt atgactttgg agatttcatt cagaccacgc 961 ccgggtccgg agtacctgtc gttgactctt actactcact tctgatgcca atcctaacct 1021 taacccgagc tctcacagct gagagtcacg tggacaccga cctcacaaaa ccgtatatca 1081 agtgggattt gctgaagtac gatttcacgg aagagaggtt aaagctcttt gatcgttatt 1141 tcaaatactg ggaccagact tatcatccca actgtgtgaa ttgtctggac gacagatgca 1201 tacttcattg tgccaatttc aatgtactgt ttagcacagt gttcccacca acttcgtttg 1261 gtcctctagt gcgcaaaatc tttgtggatg gggtgccctt cgttgtttcc acaggctatc 1321 actttcgtga gctcggcgta gttcacaacc aagatgtcaa cctgcattct tccaggttaa 1381 gttttaagga acttctggtg tatgccgcag accctgcgat gcacgctgca agcggtaatt 1441 tgttgctgga taagcggacc acgtgcttca gcgtggcggc tttaacaaat aacgttgcct 1501 tccaaaccgt caagcctggc aacttcaata aagatttcta cgatttcgcc gtcagtaaag 1561 ggttttttaa ggagggatca agcgtagaat tgaaacattt tttctttgct caggacggta 1621 atgccgctat ctcggactac gactactacc ggtataacct accaactatg tgcgacattc 1681 ggcagctgtt gttcgtggtg gaggtcgttg ataaatactt tgattgctac gatggcggat 1741 gcattaacgc caatcaagtc atcgtgaaca atctggacaa gagcgccggc ttccccttta 1801 acaaatgggg aaaggcaagg ctatattatg actctatgtc ctatgaggat caggacgctc 1861 tcttcgcgta taccaagcgc aacgttatcc ccacaattac ccagatgaac ctcaaatatg 1921 caatcagcgc aaagaacaga gcgaggacag ttgctggggt gagtatatgt tcgacaatga 1981 ctaacaggca gtttcaccag aagctgttga aatctattgc cgctacacga ggggctaccg 2041 tcgtgattgg cacctccaaa ttctacggag ggtggcataa tatgttaaaa actgtgtact 2101 ccgacgtgga aaaccctcac ctgatgggtt gggattatcc taagtgcgac cgggcgatgc 2161 caaacatgct gagaattatg gcgtcattag tcctggcgcg aaagcacacc acgtgctgta 2221 gtctgtcaca tcgcttttac agattggcta atgaatgcgc ccaagtgctt agcgagatgg 2281 tgatgtgtgg gggttcactt tacgtgaagc ccggaggaac gtcctcaggc gacgccacaa 2341 ctgcctacgc aaattcagta ttcaacatat gtcaggccgt tacagcaaac gtgaatgctc 2401 tgctgtctac tgatgggaac aagatcgccg ataagtacgt ccggaacctg caacacaggc 2461 tgtacgagtg tctgtatcgt aatcgagatg tagacaccga cttcgtgaac gaattttacg 2521 catacttacg gaaacatttc tccatgatga tcctctccga cgatgcagtt gtgtgcttta 2581 attcaactta cgcctctcag ggcttggtgg ccagtatcaa aaattttaag tcggttcttt 2641 attatcagaa taacgtgttt atgagcgagg ctaagtgttg gaccgagact gatctgacca 2701 aaggcccgca cgagttctgt tcccagcaca ccatgctggt caagcagggt gatgactatg 2761 tctatttacc atacccagat ccgagtagga tcttgggagc cggttgtttt gtggatgaca 2821 ttgtgaagac tgacggtaca ctcatgattg agcgcttcgt cagtttggca atcgacgctt 2881 acccccttac caaacatcca aatcaggaat atgcggatgt gtttcacctg taccttcaat 2941 acataaggaa acttcatgat gaactaactg gacatatgct cgacatgtat tctgtaatgc 3001 ttacaaatga caatacctca cggtactggg aaccggagtt ttatgaagcc atgtacaccc 3061 cacatactgt cctgcaagaa aacctttact ttcagtcgat ggattataag gaccacgatg 3121 gagactacaa agatcatgac attgattaca aggacgacga tgacaaataa gctagcttga 3181 ctgactgaga tacagcgtac cttcagctca cagacatgat aagatacatt gatgagtttg 3241 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 3301 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc 3361 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct 3421 acaaatgtgg tattggccca tctctatcgg tatcgtagca taaccccttg gggcctctaa 3481 acgggtcttg aggggttttt tgtgcccctc gggccggatt gctatctacc ggcattggcg 3541 cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag 3601 caacggatac ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt 3661 atccttaagg tcgtcagcta tcctgcaggc gatctctcga tttcgatcaa gacattcctt 3721 taatggtctt ttctggacac cactaggggt cagaagtagt tcatcaaact ttcttccctc 3781 cctaatctca ttggttacct tgggctatcg aaacttaatt aaccagtcaa gtcagctact 3841 tggcgagatc gacttgtctg ggtttcgact acgctcagaa ttgcgtcagt caagttcgat 3901 ctggtccttg ctattgcacc cgttctccga ttacgagttt catttaaatc atgtgagcaa 3961 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 4021 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 4081 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 4141 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 4201 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 4261 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 4321 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 4381 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 4441 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 4501 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 4561 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 4621 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 4681 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 4741 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 4801 cagcgatctg tctatttcgt tcatccatag ttgcatttaa atttccgaac tctccaaggc 4861 cctcgtcgga aaatcttcaa acctttcgtc cgatccatct tgcaggctac ctctcgaacg 4921 aactatcgca agtctcttgg ccggccttgc gccttggcta ttgcttggca gcgcctatcg 4981 ccaggtatta ctccaatccc gaatatccga gatcgggatc acccgagaga agttcaacct 5041 acatcctcaa tcccgatcta tccgagatcc gaggaatatc gaaatcgggg cgcgcctggc 5101 ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct cgtcacggcg agcgctgcca 5161 cgtcagacga agggcgcagg agcgtcctga tccttccgcc cggacgctca ggacagcggc 5221 ccgctgctca taagactcgg ccttagaacc ccagtatcag cagaaggaca ttttaggacg 5281 ggacttgggt gactctaggg cactggtttt ctttccagag agcggaacag gcgaggaaaa 5341 gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac gccgatgatt 5401 atataaggac gcgccgggtg tggcacagct agttccgtcg cagccgggat ttgggtcgcg 5461 gttcttgttt gtggatcgct gtgatcgtca cttggtgagt agcgggctgc tgggctggcc 5521 ggggctttcg tggccgccgg gccgctcggt gggacggaag cgtgtggaga gaccgccaag 5581 ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg ggggttgggg ggagcgcagc 5641 aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg ctgtgaggtc 5701 gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc aaggtcttga gcccttcgct 5761 aatgcgggaa agctcttatt cgggtgagat gggctgggca ccatctgggg accctgacgt 5821 gaagtttgtc actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg 5881 cggtgccgtt gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac 5941 gtcacccgtt ctgttggctt ataatgcagg gtggggccac ctgccggtag gtgtgcggta 6001 ggcttttctc cgtcgcagga cgcagggttc gggcctaggg taggctctcc tgaatcgaca 6061 ggcgccggac ctctggtgag gggagggata agtgaggcgt cagtttcttt ggtcggtttt 6121 atgtacctat cttcttaagt agctgaagct ccggttttga actatgcgct cggggttggc 6181 gagtgtgttt tgtgaagttt tttaggcacc ttttgaaatg taatcatttg ggtcaatatg 6241 taattttcag tgttagactt gtaaattgtc cgctaaattc tggccgtttt tggctttttt 6301 gttagacaac atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc 6361 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt 6421 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt 6481 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac 6541 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga 6601 gttgagcggt tcccggctgg ccgcgcagca acagatggag ggcctcctgg cgccgcaccg 6661 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa 6721 gggtctgggc agcgccgtcg tgctccccgg agtggaggcc gccgagcgcg ccggggtgcc 6781 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac 6841 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc 6901 cggtgcctag gctagtatgt aagcctagtc ttagataata aaatcgctat ccatcgaaga 6961 tggatgtgtg ttggtttttt gtgtgtgtaa cgctaggcgc gcctggtgta ccgagaacga 7021 tcctctcagt gcgagtctcg acgatccata tcgttgcttg gcagtcagcc agtcggaatc 7081 cagcttggga cccaggaagt ccaatcgtca gatattgtac tcaagcctgg tcacggcagc 7141 gtaccgatct gtttaaacct agatattgat agtctgatcg gtcaacgtat aatcgagtcc 7201 tagcttttgc aaacatctat caagagacag gatcagcagg aggctttcgc atgagtattc 7261 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 7321 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgcg cgagtgggtt 7381 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgct 7441 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 7501 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtatt 7561 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 7621 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatt ggaggaccga 7681 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 7741 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 7801 tggcaacaac cttgcgtaaa ctattaactg gcgaactact tactctagct tcccggcaac 7861 agttgataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 7921 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 7981 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 8041 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 8101 agcattggta accgattcta ggtgcattgg cgcagaaaaa aatgcctgat gcgacgctgc 8161 gcgtcttata ctcccacata tgccagattc agcaacggat acggcttccc caacttgccc 8221 acttccatac gtgtcctcct taccagaaat ttatccttaa gatcccgaat cgtttaaact 8281 cgactctggc tctatcgaat ctccgtcgtt tcgagcttac gcgaacagcc gtggcgctca 8341 tttgctcgtc gggcatcgaa tctcgtcagc tatcgtcagc ttaccttttt ggcagcgatc 8401 gcggctcccg acatcttgga ccattagctc cacaggtatc ttcttccctc tagtggtcat 8461 aacagcagct tcagctacct ctcaattcaa aaaacccctc aagacccgtt tagaggcccc 8521 aaggggttat gctatcaatc gttgcgttac acacacaaaa aaccaacaca catccatctt 8581 cgatggatag cgattttatt atctaactgc tgatcgagtg tagccagatc tagtaatcaa 8641 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 8701 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 8761 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 8821 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 8881 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 8941 ctacttggca gtacatctac gtattagtca tcgctattac catg //