LOCUS Exported 8305 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 helicase. ACCESSION . VERSION . KEYWORDS pGBW-m4133394 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8305) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 8305) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8305 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1811..1831 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 2180..2197 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 2274..2395 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2285..2304) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2339..2358 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 2510..2556 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 2586..2722 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2767..2838 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(3064..3652) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3144..3163) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 4152..5361 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 5198..5217 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 5365..5964 /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(5365..5384) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5861..5881 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5991..6039 /note="synthetic polyadenylation signal" CDS 6305..7165 /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(6523..6542) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 7178..7314 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 7459..7530 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 7535..7581 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7599..7647 /note="synthetic polyadenylation signal" enhancer 7728..8031 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 8033..8235 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 8186..8206 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 8304..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatggcg gtgggggctt gcgtactatg taacagccag acgtcattgc gctgcggtgc 61 atgtataaga aggccgtttc tgtgttgcaa atgctgctac gatcatgtta tttcgacttc 121 tcacaagctt gtgctcagtg tcaatcctta tgtgtgtaat gcccccggat gcgacgtcac 181 agatgttacc caactgtacc tgggcggcat gtcctattat tgtaagagcc acaaaccacc 241 aatctccttc cccctgtgcg ccaacggaca ggtgtttggg ctctacaaaa acacatgtgt 301 gggatctgac aatgtcaccg acttcaacgc tatcgccaca tgcgattgga ctaatgcagg 361 cgactacatt ttagccaaca cctgtaccga acggctgaag ttatttgcgg ctgagactct 421 aaaggcaact gaggaaacct tcaaactttc atatggtatc gccacggtac gtgaggtgtt 481 gtctgatcga gaactgcatc tcagttggga ggtcgggaag cctagacctc cccttaatcg 541 aaattacgtg ttcacaggtt atagggttac caagaacagc aaagtccaga tcggcgaata 601 tacttttgag aagggagatt acggtgacgc cgtagtgtat cgtggcacaa caacatacaa 661 actgaatgtt ggggattact tcgtgctgac gtcccacacc gttatgccgc tctcagctcc 721 aactttggtt ccccaggaac attacgtgcg gattactgga ctctatccaa cgctaaacat 781 aagcgacgag ttttccagca acgtggctaa ttaccaaaag gtcggcatgc agaagtactc 841 cacccttcag ggtcctcctg ggactggcaa aagtcacttc gccatcggac tggcacttta 901 ctatcccagt gcgcgcattg tatataccgc ctgctcacat gcggcagtgg acgccctgtg 961 tgagaaggct ttaaagtatc tgccaataga caaatgctct cgcattatcc ctgccagggc 1021 cagagtggag tgctttgata agtttaaagt gaactcgacc ctcgaacagt atgtcttctg 1081 cacagttaac gcattgcccg aaacgaccgc cgatatagtt gtgttcgacg aaatctcaat 1141 ggctaccaat tatgatctct ccgtcgtcaa tgcacggctg cgggccaaac actacgtcta 1201 catcggggac cccgcgcagt taccagcacc gcgaacccta ctgactaagg gcacacttga 1261 gccagagtac ttcaacagcg tatgtagact gatgaaaacc attggccctg atatgttcct 1321 gggaacatgt cggaggtgcc ccgctgaaat agtggacacc gtttcagcac ttgtgtacga 1381 caataagctc aaagcacaca aggataaatc cgcacaatgt tttaagatgt tttacaaagg 1441 ggttattacc catgatgtgt ctagcgctat caatcgccct cagattggcg tggtgagaga 1501 gtttttgacg cgaaatcccg cctggcgaaa ggccgtattc atcagtcctt acaactcgca 1561 aaatgccgtc gctagtaaga ttttaggact ccctactcag acagttgata gctctcaagg 1621 ttctgaatac gattatgtga tttttactca gactaccgaa acggcacatt cttgtaacgt 1681 caaccgtttt aacgtggcca ttacaagggc caaggttggc atcttgtgta tcatgtcaga 1741 ccgggacctg tatgataaac tgcaattcac atctttggag ataccgcgcc gcaatgtcgc 1801 tacccttcag gaaaaccttt actttcagtc gatgagtgac aaaatcattc atctaaccga 1861 tgactccttt gatacagacg tgttgaaggc cgatggtgcg atactggtag atttctgggc 1921 tgaatggtgt ggcccgtgca agatgatcgc acccattctg gacgagatcg ccgacgagta 1981 tcaaggaaaa ttaactgtcg ctaagcttaa tatagatcag aacccaggga cggcacctaa 2041 atacggcatc aggggaatac ctaccctcct gctctttaag aatggggaag ttgccgcgac 2101 aaaagtggga gccctgtcta agggccagct aaaagagttc ttggacgcta acctggcagg 2161 ttcaggctcg gggcacatgc accatcacca tcaccatagc agcgggctcg tgccacgcgg 2221 ctaagctagc ttgactgact gagatacagc gtaccttcag ctcacagaca tgataagata 2281 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 2341 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 2401 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag 2461 caagtaaaac ctctacaaat gtggtattgg cccatctcta tcggtatcgt agcataaccc 2521 cttggggcct ctaaacgggt cttgaggggt tttttgtgcc cctcgggccg gattgctatc 2581 taccggcatt ggcgcagaaa aaaatgcctg atgcgacgct gcgcgtctta tactcccaca 2641 tatgccagat tcagcaacgg atacggcttc cccaacttgc ccacttccat acgtgtcctc 2701 cttaccagaa atttatcctt aaggtcgtca gctatcctgc aggcgatctc tcgatttcga 2761 tcaagacatt cctttaatgg tcttttctgg acaccactag gggtcagaag tagttcatca 2821 aactttcttc cctccctaat ctcattggtt accttgggct atcgaaactt aattaaccag 2881 tcaagtcagc tacttggcga gatcgacttg tctgggtttc gactacgctc agaattgcgt 2941 cagtcaagtt cgatctggtc cttgctattg cacccgttct ccgattacga gtttcattta 3001 aatcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 3061 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 3121 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 3181 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 3241 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 3301 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 3361 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 3421 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 3481 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 3541 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 3601 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 3661 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 3721 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 3781 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 3841 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcat ttaaatttcc 3901 gaactctcca aggccctcgt cggaaaatct tcaaaccttt cgtccgatcc atcttgcagg 3961 ctacctctcg aacgaactat cgcaagtctc ttggccggcc ttgcgccttg gctattgctt 4021 ggcagcgcct atcgccaggt attactccaa tcccgaatat ccgagatcgg gatcacccga 4081 gagaagttca acctacatcc tcaatcccga tctatccgag atccgaggaa tatcgaaatc 4141 ggggcgcgcc tggcctccgc gccgggtttt ggcgcctccc gcgggcgccc ccctcgtcac 4201 ggcgagcgct gccacgtcag acgaagggcg caggagcgtc ctgatccttc cgcccggacg 4261 ctcaggacag cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag 4321 gacattttag gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga 4381 acaggcgagg aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt 4441 gaacgccgat gattatataa ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg 4501 ggatttgggt cgcggttctt gtttgtggat cgctgtgatc gtcacttggt gagtagcggg 4561 ctgctgggct ggccggggct ttcgtggccg ccgggccgct cggtgggacg gaagcgtgtg 4621 gagagaccgc caagggctgt agtctgggtc cgcgagcaag gttgccctga actgggggtt 4681 ggggggagcg cagcaaaatg gcggctgttc ccgagtcttg aatggaagac gcttgtgagg 4741 cgggctgtga ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga acccaaggtc 4801 ttgagccctt cgctaatgcg ggaaagctct tattcgggtg agatgggctg ggcaccatct 4861 ggggaccctg acgtgaagtt tgtcactgac tggagaactc ggtttgtcgt ctgttgcggg 4921 ggcggcagtt atggcggtgc cgttgggcag tgcacccgta cctttgggag cgcgcgccct 4981 cgtcgtgtcg tgacgtcacc cgttctgttg gcttataatg cagggtgggg ccacctgccg 5041 gtaggtgtgc ggtaggcttt tctccgtcgc aggacgcagg gttcgggcct agggtaggct 5101 ctcctgaatc gacaggcgcc ggacctctgg tgaggggagg gataagtgag gcgtcagttt 5161 ctttggtcgg ttttatgtac ctatcttctt aagtagctga agctccggtt ttgaactatg 5221 cgctcggggt tggcgagtgt gttttgtgaa gttttttagg caccttttga aatgtaatca 5281 tttgggtcaa tatgtaattt tcagtgttag acttgtaaat tgtccgctaa attctggccg 5341 tttttggctt ttttgttaga caacatgacc gagtacaagc ccacggtgcg cctcgccacc 5401 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc 5461 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc 5521 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg 5581 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc 5641 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggagggcctc 5701 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc 5761 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggccgccgag 5821 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag 5881 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc 5941 atgacccgca agcccggtgc ctaggctagt atgtaagcct agtcttagat aataaaatcg 6001 ctatccatcg aagatggatg tgtgttggtt ttttgtgtgt gtaacgctag gcgcgcctgg 6061 tgtaccgaga acgatcctct cagtgcgagt ctcgacgatc catatcgttg cttggcagtc 6121 agccagtcgg aatccagctt gggacccagg aagtccaatc gtcagatatt gtactcaagc 6181 ctggtcacgg cagcgtaccg atctgtttaa acctagatat tgatagtctg atcggtcaac 6241 gtataatcga gtcctagctt ttgcaaacat ctatcaagag acaggatcag caggaggctt 6301 tcgcatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 6361 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 6421 tgcgcgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 6481 ccccgaagaa cgctttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 6541 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 6601 cttggttgag tattcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 6661 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 6721 gattggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 6781 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 6841 gatgcctgta gcaatggcaa caaccttgcg taaactatta actggcgaac tacttactct 6901 agcttcccgg caacagttga tagactggat ggaggcggat aaagttgcag gaccacttct 6961 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 7021 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 7081 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 7141 tgcctcactg attaagcatt ggtaaccgat tctaggtgca ttggcgcaga aaaaaatgcc 7201 tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac ggatacggct 7261 tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc ttaagatcgt 7321 ttaaactcga ctctggctct atcgaatctc cgtcgtttcg agcttacgcg aacagccgtg 7381 gcgctcattt gctcgtcggg catcgaatct cgtcagctat cgtcagctta cctttttggc 7441 agcgatcgcg gctcccgaca tcttggacca ttagctccac aggtatcttc ttccctctag 7501 tggtcataac agcagcttca gctacctctc aattcaaaaa acccctcaag acccgtttag 7561 aggccccaag gggttatgct atcaatcgtt gcgttacaca cacaaaaaac caacacacat 7621 ccatcttcga tggatagcga ttttattatc taactgctga tcgagtgtag ccagatctag 7681 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 7741 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 7801 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 7861 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 7921 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 7981 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat gctgatgcgg 8041 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 8101 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 8161 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 8221 tatataagca gagctggttt agtgaaccgt cagatcagat ctttgtcgat cctaccatcc 8281 actcgacaca cccgccagcg gccgc //