LOCUS Exported 7985 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 helicase. ACCESSION . VERSION . KEYWORDS pGBW-m4134443 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7985) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7985) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7985 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..304 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 306..508 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 459..479 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 577..586 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2383..2403 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 2407..2472 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 2525..2646 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2536..2555) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2590..2609 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 2761..2807 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 2837..2973 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 3018..3089 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(3315..3903) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3395..3414) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 4403..5612 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 5449..5468 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 5616..6215 /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(5616..5635) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 6112..6132 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 6242..6290 /note="synthetic polyadenylation signal" CDS 6556..7416 /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(6774..6793) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 7429..7565 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 7717..7788 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 7793..7839 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 7857..7905 /note="synthetic polyadenylation signal" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 301 catgctgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatca gatctttgtc 541 gatcctacca tccactcgac acacccgcca gcggccgcca ccatggttgg ggcttgtgtt 601 ctttgcaatt cacagacttc attaagatgt ggtgcttgca tacgtagacc attcttatgt 661 tgtaaatgct gttacgacca tgtcatatca acatcacata aattagtctt gtctgttaat 721 ccgtatgttt gcaatgctcc aggttgtgat gtcacagatg tgactcaact ttacttagga 781 ggtatgagct attattgtaa atcacataaa ccacccatta gttttccatt gtgtgctaat 841 ggacaagttt ttggtttata taaaaataca tgtgttggta gcgataatgt tactgacttt 901 aatgcaattg caacatgtga ctggacaaat gctggtgatt acattttagc taacacctgt 961 actgaaagac tcaagctttt tgcagcagaa acgctcaaag ctactgagga gacatttaaa 1021 ctgtcttatg gtattgctac tgtacgtgaa gtgctgtctg acagagaatt acatctttca 1081 tgggaagttg gtaaacctag accaccactt aaccgaaatt atgtctttac tggttatcgt 1141 gtaactaaaa acagtaaagt acaaatagga gagtacacct ttgaaaaagg tgactatggt 1201 gatgctgttg tttaccgagg tacaacaact tacaaattaa atgttggtga ttattttgtg 1261 ctgacatcac atacagtaat gccattaagt gcacctacac tagtgccaca agagcactat 1321 gttagaatta ctggcttata cccaacactc aatatctcag atgagttttc tagcaatgtt 1381 gcaaattatc aaaaggttgg tatgcaaaag tattctacac tccagggacc acctggtact 1441 ggtaagagtc attttgctat tggcctagct ctctactacc cttctgctcg catagtgtat 1501 acagcttgct ctcatgccgc tgttgatgca ctatgtgaga aggcattaaa atatttgcct 1561 atagataaat gtagtagaat tatacctgca cgtgctcgtg tagagtgttt tgataaattc 1621 aaagtgaatt caacattaga acagtatgtc ttttgtactg taaatgcatt gcctgagacg 1681 acagcagata tagttgtctt tgatgaaatt tcaatggcca caaattatga tttgagtgtt 1741 gtcaatgcca gattacgtgc taagcactat gtgtacattg gcgaccctgc tcaattacct 1801 gcaccacgca cattgctaac taagggcaca ctagaaccag aatatttcaa ttcagtgtgt 1861 agacttatga aaactatagg tccagacatg ttcctcggaa cttgtcggcg ttgtcctgct 1921 gaaattgttg acactgtgag tgctttggtt tatgataata agcttaaagc acataaagac 1981 aaatcagctc aatgctttaa aatgttttat aagggtgtta tcacgcatga tgtttcatct 2041 gcaattaaca ggccacaaat aggcgtggta agagaattcc ttacacgtaa ccctgcttgg 2101 agaaaagctg tctttatttc accttataat tcacagaatg ctgtagcctc aaagattttg 2161 ggactaccaa ctcaaactgt tgattcatca cagggctcag aatatgacta tgtcatattc 2221 actcaaacca ctgaaacagc tcactcttgt aatgtaaaca gatttaatgt tgctattacc 2281 agagcaaaag taggcatact ttgcataatg tctgatagag acctttatga caagttgcaa 2341 tttacaagtc ttgaaattcc acgtaggaat gtggcaactt tagaaaacct ttactttcag 2401 tcgatggatt ataaggacca cgatggagac tacaaagatc atgacattga ttacaaggac 2461 gacgatgaca aataagctag cttgactgac tgagatacag cgtaccttca gctcacagac 2521 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 2581 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 2641 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag 2701 gttttttaaa gcaagtaaaa cctctacaaa tgtggtattg gcccatctct atcggtatcg 2761 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgtgc ccctcgggcc 2821 ggattgctat ctaccggcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt 2881 atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca 2941 tacgtgtcct ccttaccaga aatttatcct taaggtcgtc agctatcctg caggcgatct 3001 ctcgatttcg atcaagacat tcctttaatg gtcttttctg gacaccacta ggggtcagaa 3061 gtagttcatc aaactttctt ccctccctaa tctcattggt taccttgggc tatcgaaact 3121 taattaacca gtcaagtcag ctacttggcg agatcgactt gtctgggttt cgactacgct 3181 cagaattgcg tcagtcaagt tcgatctggt ccttgctatt gcacccgttc tccgattacg 3241 agtttcattt aaatcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 3301 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 3361 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 3421 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 3481 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 3541 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 3601 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 3661 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 3721 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 3781 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 3841 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 3901 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 3961 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 4021 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 4081 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgca 4141 tttaaatttc cgaactctcc aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc 4201 catcttgcag gctacctctc gaacgaacta tcgcaagtct cttggccggc cttgcgcctt 4261 ggctattgct tggcagcgcc tatcgccagg tattactcca atcccgaata tccgagatcg 4321 ggatcacccg agagaagttc aacctacatc ctcaatcccg atctatccga gatccgagga 4381 atatcgaaat cggggcgcgc ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc 4441 cccctcgtca cggcgagcgc tgccacgtca gacgaagggc gcaggagcgt cctgatcctt 4501 ccgcccggac gctcaggaca gcggcccgct gctcataaga ctcggcctta gaaccccagt 4561 atcagcagaa ggacatttta ggacgggact tgggtgactc tagggcactg gttttctttc 4621 cagagagcgg aacaggcgag gaaaagtagt cccttctcgg cgattctgcg gagggatctc 4681 cgtggggcgg tgaacgccga tgattatata aggacgcgcc gggtgtggca cagctagttc 4741 cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg 4801 tgagtagcgg gctgctgggc tggccggggc tttcgtggcc gccgggccgc tcggtgggac 4861 ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg 4921 aactgggggt tggggggagc gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga 4981 cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt gggcggcaag 5041 aacccaaggt cttgagccct tcgctaatgc gggaaagctc ttattcgggt gagatgggct 5101 gggcaccatc tggggaccct gacgtgaagt ttgtcactga ctggagaact cggtttgtcg 5161 tctgttgcgg gggcggcagt tatggcggtg ccgttgggca gtgcacccgt acctttggga 5221 gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt ggcttataat gcagggtggg 5281 gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg caggacgcag ggttcgggcc 5341 tagggtaggc tctcctgaat cgacaggcgc cggacctctg gtgaggggag ggataagtga 5401 ggcgtcagtt tctttggtcg gttttatgta cctatcttct taagtagctg aagctccggt 5461 tttgaactat gcgctcgggg ttggcgagtg tgttttgtga agttttttag gcaccttttg 5521 aaatgtaatc atttgggtca atatgtaatt ttcagtgtta gacttgtaaa ttgtccgcta 5581 aattctggcc gtttttggct tttttgttag acaacatgac cgagtacaag cccacggtgc 5641 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg 5701 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc 5761 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg 5821 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg 5881 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga 5941 tggagggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg 6001 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg 6061 aggccgccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc 6121 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc 6181 gcacctggtg catgacccgc aagcccggtg cctaggctag tatgtaagcc tagtcttaga 6241 taataaaatc gctatccatc gaagatggat gtgtgttggt tttttgtgtg tgtaacgcta 6301 ggcgcgcctg gtgtaccgag aacgatcctc tcagtgcgag tctcgacgat ccatatcgtt 6361 gcttggcagt cagccagtcg gaatccagct tgggacccag gaagtccaat cgtcagatat 6421 tgtactcaag cctggtcacg gcagcgtacc gatctgttta aacctagata ttgatagtct 6481 gatcggtcaa cgtataatcg agtcctagct tttgcaaaca tctatcaaga gacaggatca 6541 gcaggaggct ttcgcatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 6601 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 6661 gatcagttgg gtgcgcgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 6721 gagagttttc gccccgaaga acgctttcca atgatgagca cttttaaagt tctgctatgt 6781 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 6841 tctcagaatg acttggttga gtattcacca gtcacagaaa agcatcttac ggatggcatg 6901 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 6961 cttctgacaa cgattggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 7021 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 7081 cgtgacacca cgatgcctgt agcaatggca acaaccttgc gtaaactatt aactggcgaa 7141 ctacttactc tagcttcccg gcaacagttg atagactgga tggaggcgga taaagttgca 7201 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 7261 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 7321 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 7381 gctgagatag gtgcctcact gattaagcat tggtaaccga ttctaggtgc attggcgcag 7441 aaaaaaatgc ctgatgcgac gctgcgcgtc ttatactccc acatatgcca gattcagcaa 7501 cggatacggc ttccccaact tgcccacttc catacgtgtc ctccttacca gaaatttatc 7561 cttaagatcc cgaatcgttt aaactcgact ctggctctat cgaatctccg tcgtttcgag 7621 cttacgcgaa cagccgtggc gctcatttgc tcgtcgggca tcgaatctcg tcagctatcg 7681 tcagcttacc tttttggcag cgatcgcggc tcccgacatc ttggaccatt agctccacag 7741 gtatcttctt ccctctagtg gtcataacag cagcttcagc tacctctcaa ttcaaaaaac 7801 ccctcaagac ccgtttagag gccccaaggg gttatgctat caatcgttgc gttacacaca 7861 caaaaaacca acacacatcc atcttcgatg gatagcgatt ttattatcta actgctgatc 7921 gagtgtagcc agatctagta atcaattacg gggtcattag ttcatagccc atatatggag 7981 ttccg //