LOCUS Exported 8750 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 helicase. ACCESSION . VERSION . KEYWORDS pGBW-m4133203 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8750) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 8750) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8750 /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 2290..2411 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2301..2320) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2355..2374 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 2526..2572 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 2602..2738 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2783..2854 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(3080..3668) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3160..3179) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 4168..5377 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 5214..5233 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 5381..6409 /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 6436..6484 /note="synthetic polyadenylation signal" CDS 6750..7610 /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(6968..6987) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 7623..7759 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 7904..7975 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 7980..8026 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 8044..8092 /note="synthetic polyadenylation signal" enhancer 8173..8476 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 8478..8680 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 8631..8651 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 8749..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 ctaaggatcc gcaggcctct gctagcttga ctgactgaga tacagcgtac cttcagctca 2281 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 2341 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 2401 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 2461 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tattggccca tctctatcgg 2521 tatcgtagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgtgcccctc 2581 gggccggatt gctatctacc ggcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc 2641 gtcttatact cccacatatg ccagattcag caacggatac ggcttcccca acttgcccac 2701 ttccatacgt gtcctcctta ccagaaattt atccttaagg tcgtcagcta tcctgcaggc 2761 gatctctcga tttcgatcaa gacattcctt taatggtctt ttctggacac cactaggggt 2821 cagaagtagt tcatcaaact ttcttccctc cctaatctca ttggttacct tgggctatcg 2881 aaacttaatt aaccagtcaa gtcagctact tggcgagatc gacttgtctg ggtttcgact 2941 acgctcagaa ttgcgtcagt caagttcgat ctggtccttg ctattgcacc cgttctccga 3001 ttacgagttt catttaaatc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 3061 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 3121 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 3181 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 3241 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 3301 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 3361 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 3421 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 3481 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 3541 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 3601 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 3661 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 3721 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 3781 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 3841 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 3901 ttgcatttaa atttccgaac tctccaaggc cctcgtcgga aaatcttcaa acctttcgtc 3961 cgatccatct tgcaggctac ctctcgaacg aactatcgca agtctcttgg ccggccttgc 4021 gccttggcta ttgcttggca gcgcctatcg ccaggtatta ctccaatccc gaatatccga 4081 gatcgggatc acccgagaga agttcaacct acatcctcaa tcccgatcta tccgagatcc 4141 gaggaatatc gaaatcgggg cgcgcctggc ctccgcgccg ggttttggcg cctcccgcgg 4201 gcgcccccct cgtcacggcg agcgctgcca cgtcagacga agggcgcagg agcgtcctga 4261 tccttccgcc cggacgctca ggacagcggc ccgctgctca taagactcgg ccttagaacc 4321 ccagtatcag cagaaggaca ttttaggacg ggacttgggt gactctaggg cactggtttt 4381 ctttccagag agcggaacag gcgaggaaaa gtagtccctt ctcggcgatt ctgcggaggg 4441 atctccgtgg ggcggtgaac gccgatgatt atataaggac gcgccgggtg tggcacagct 4501 agttccgtcg cagccgggat ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtca 4561 cttggtgagt agcgggctgc tgggctggcc ggggctttcg tggccgccgg gccgctcggt 4621 gggacggaag cgtgtggaga gaccgccaag ggctgtagtc tgggtccgcg agcaaggttg 4681 ccctgaactg ggggttgggg ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatg 4741 gaagacgctt gtgaggcggg ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg 4801 gcaagaaccc aaggtcttga gcccttcgct aatgcgggaa agctcttatt cgggtgagat 4861 gggctgggca ccatctgggg accctgacgt gaagtttgtc actgactgga gaactcggtt 4921 tgtcgtctgt tgcgggggcg gcagttatgg cggtgccgtt gggcagtgca cccgtacctt 4981 tgggagcgcg cgccctcgtc gtgtcgtgac gtcacccgtt ctgttggctt ataatgcagg 5041 gtggggccac ctgccggtag gtgtgcggta ggcttttctc cgtcgcagga cgcagggttc 5101 gggcctaggg taggctctcc tgaatcgaca ggcgccggac ctctggtgag gggagggata 5161 agtgaggcgt cagtttcttt ggtcggtttt atgtacctat cttcttaagt agctgaagct 5221 ccggttttga actatgcgct cggggttggc gagtgtgttt tgtgaagttt tttaggcacc 5281 ttttgaaatg taatcatttg ggtcaatatg taattttcag tgttagactt gtaaattgtc 5341 cgctaaattc tggccgtttt tggctttttt gttagacaac atgggtaaaa agcctgaact 5401 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat 5461 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata 5521 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca 5581 ctttgcatcc gccgcgctcc cgattccgga agtgcttgac attggggagt tcagcgagag 5641 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac 5701 cgaactgccc gctgttctgc agccggtcgc ggaggcaatg gatgccatcg ctgccgccga 5761 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac 5821 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat 5881 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga 5941 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac 6001 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca 6061 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac 6121 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccccggctcc gggcgtatat 6181 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc 6241 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg 6301 tacacaaatc gcccgcagaa gcgccgccgt ctggaccgat ggctgtgtag aagtactcgc 6361 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaataag ctagtatgta 6421 agcctagtct tagataataa aatcgctatc catcgaagat ggatgtgtgt tggttttttg 6481 tgtgtgtaac gctaggcgcg cctggtgtac cgagaacgat cctctcagtg cgagtctcga 6541 cgatccatat cgttgcttgg cagtcagcca gtcggaatcc agcttgggac ccaggaagtc 6601 caatcgtcag atattgtact caagcctggt cacggcagcg taccgatctg tttaaaccta 6661 gatattgata gtctgatcgg tcaacgtata atcgagtcct agcttttgca aacatctatc 6721 aagagacagg atcagcagga ggctttcgca tgagtattca acatttccgt gtcgccctta 6781 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 6841 taaaagatgc tgaagatcag ttgggtgcgc gagtgggtta catcgaactg gatctcaaca 6901 gcggtaagat ccttgagagt tttcgccccg aagaacgctt tccaatgatg agcactttta 6961 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 7021 gccgcataca ctattctcag aatgacttgg ttgagtattc accagtcaca gaaaagcatc 7081 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 7141 ctgcggccaa cttacttctg acaacgattg gaggaccgaa ggagctaacc gcttttttgc 7201 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 7261 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacc ttgcgtaaac 7321 tattaactgg cgaactactt actctagctt cccggcaaca gttgatagac tggatggagg 7381 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 7441 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 7501 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 7561 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ccgattctag 7621 gtgcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac tcccacatat 7681 gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg tgtcctcctt 7741 accagaaatt tatccttaag atcgtttaaa ctcgactctg gctctatcga atctccgtcg 7801 tttcgagctt acgcgaacag ccgtggcgct catttgctcg tcgggcatcg aatctcgtca 7861 gctatcgtca gcttaccttt ttggcagcga tcgcggctcc cgacatcttg gaccattagc 7921 tccacaggta tcttcttccc tctagtggtc ataacagcag cttcagctac ctctcaattc 7981 aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt atgctatcaa tcgttgcgtt 8041 acacacacaa aaaaccaaca cacatccatc ttcgatggat agcgatttta ttatctaact 8101 gctgatcgag tgtagccaga tctagtaatc aattacgggg tcattagttc atagcccata 8161 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 8221 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 8281 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 8341 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 8401 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 8461 catcgctatt accatgctga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 8521 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 8581 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 8641 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat 8701 cagatctttg tcgatcctac catccactcg acacacccgc cagcggccgc //