LOCUS Exported 7651 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp4. ACCESSION . VERSION . KEYWORDS pGBW-m4133795 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7651) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7651) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7651 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1502..1522 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1523..1567 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" polyA_signal 1620..1741 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1631..1650) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1685..1704 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1856..1902 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1932..2068 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2113..2184 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2410..2998) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2490..2509) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3498..4707 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4544..4563 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4711..5310 /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(4711..4730) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5207..5227 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5337..5385 /note="synthetic polyadenylation signal" CDS 5651..6511 /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(5869..5888) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6524..6660 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6805..6876 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6881..6927 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6945..6993 /note="synthetic polyadenylation signal" enhancer 7074..7377 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 7379..7581 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 7532..7552 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" ORIGIN 1 caccatgatt gttaataatt ggttgaagca gttaattaaa gttacacttg tgttcctttt 61 tgttgctgct attttctatt taataacacc tgttcatgtc atgtctaaac atactgactt 121 ttcaagtgaa atcataggat acaaggctat tgatggtggt gtcactcgtg acatagcatc 181 tacagatact tgttttgcta acaaacatgc tgattttgac acatggttta gccagcgtgg 241 tggtagttat actaatgaca aagcttgccc attgattgct gcagtcataa caagagaagt 301 gggttttgtc gtgcctggtt tgcctggcac gatattacgc acaactaatg gtgacttttt 361 gcatttctta cctagagttt ttagtgcagt tggtaacatc tgttacacac catcaaaact 421 tatagagtac actgactttg caacatcagc ttgtgttttg gctgctgaat gtacaatttt 481 taaagatgct tctggtaagc cagtaccata ttgttatgat accaatgtac tagaaggttc 541 tgttgcttat gaaagtttac gccctgacac acgttatgtg ctcatggatg gctctattat 601 tcaatttcct aacacctacc ttgaaggttc tgttagagtg gtaacaactt ttgattctga 661 gtactgtagg cacggcactt gtgaaagatc agaagctggt gtttgtgtat ctactagtgg 721 tagatgggta cttaacaatg attattacag atctttacca ggagttttct gtggtgtaga 781 tgctgtaaat ttacttacta atatgtttac accactaatt caacctattg gtgctttgga 841 catatcagca tctatagtag ctggtggtat tgtagctatc gtagtaacat gccttgccta 901 ctattttatg aggtttagaa gagcttttgg tgaatacagt catgtagttg cctttaatac 961 tttactattc cttatgtcat tcactgtact ctgtttaaca ccagtttact cattcttacc 1021 tggtgtttat tctgttattt acttgtactt gacattttat cttactaatg atgtttcttt 1081 tttagcacat attcagtgga tggttatgtt cacaccttta gtacctttct ggataacaat 1141 tgcttatatc atttgtattt ccacaaagca tttctattgg ttctttagta attacctaaa 1201 gagacgtgta gtctttaatg gtgtttcctt tagtactttt gaagaagctg cgctgtgcac 1261 ctttttgtta aataaagaaa tgtatctaaa gttgcgtagt gatgtgctat tacctcttac 1321 gcaatataat agatacttag ctctttataa taagtacaag tattttagtg gagcaatgga 1381 tacaactagc tacagagaag ctgcttgttg tcatctcgca aaggctctca atgacttcag 1441 taactcaggt tctgatgttc tttaccaacc accacaaacc tctatcacct cagctgtttt 1501 ggaaaacctt tactttcagt cgaaagaaac cgcggcagct aagttcgaga ggcagcacat 1561 ggacagttaa gctagcttga ctgactgaga tacagcgtac cttcagctca cagacatgat 1621 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat 1681 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt 1741 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt 1801 ttaaagcaag taaaacctct acaaatgtgg tattggccca tctctatcgg tatcgtagca 1861 taaccccttg gggcctctaa acgggtcttg aggggttttt tgtgcccctc gggccggatt 1921 gctatctacc ggcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact 1981 cccacatatg ccagattcag caacggatac ggcttcccca acttgcccac ttccatacgt 2041 gtcctcctta ccagaaattt atccttaagg tcgtcagcta tcctgcaggc gatctctcga 2101 tttcgatcaa gacattcctt taatggtctt ttctggacac cactaggggt cagaagtagt 2161 tcatcaaact ttcttccctc cctaatctca ttggttacct tgggctatcg aaacttaatt 2221 aaccagtcaa gtcagctact tggcgagatc gacttgtctg ggtttcgact acgctcagaa 2281 ttgcgtcagt caagttcgat ctggtccttg ctattgcacc cgttctccga ttacgagttt 2341 catttaaatc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 2401 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 2461 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 2521 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 2581 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 2641 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 2701 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 2761 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 2821 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 2881 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 2941 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 3001 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 3061 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 3121 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 3181 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcatttaa 3241 atttccgaac tctccaaggc cctcgtcgga aaatcttcaa acctttcgtc cgatccatct 3301 tgcaggctac ctctcgaacg aactatcgca agtctcttgg ccggccttgc gccttggcta 3361 ttgcttggca gcgcctatcg ccaggtatta ctccaatccc gaatatccga gatcgggatc 3421 acccgagaga agttcaacct acatcctcaa tcccgatcta tccgagatcc gaggaatatc 3481 gaaatcgggg cgcgcctggc ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct 3541 cgtcacggcg agcgctgcca cgtcagacga agggcgcagg agcgtcctga tccttccgcc 3601 cggacgctca ggacagcggc ccgctgctca taagactcgg ccttagaacc ccagtatcag 3661 cagaaggaca ttttaggacg ggacttgggt gactctaggg cactggtttt ctttccagag 3721 agcggaacag gcgaggaaaa gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg 3781 ggcggtgaac gccgatgatt atataaggac gcgccgggtg tggcacagct agttccgtcg 3841 cagccgggat ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtca cttggtgagt 3901 agcgggctgc tgggctggcc ggggctttcg tggccgccgg gccgctcggt gggacggaag 3961 cgtgtggaga gaccgccaag ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg 4021 ggggttgggg ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt 4081 gtgaggcggg ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc 4141 aaggtcttga gcccttcgct aatgcgggaa agctcttatt cgggtgagat gggctgggca 4201 ccatctgggg accctgacgt gaagtttgtc actgactgga gaactcggtt tgtcgtctgt 4261 tgcgggggcg gcagttatgg cggtgccgtt gggcagtgca cccgtacctt tgggagcgcg 4321 cgccctcgtc gtgtcgtgac gtcacccgtt ctgttggctt ataatgcagg gtggggccac 4381 ctgccggtag gtgtgcggta ggcttttctc cgtcgcagga cgcagggttc gggcctaggg 4441 taggctctcc tgaatcgaca ggcgccggac ctctggtgag gggagggata agtgaggcgt 4501 cagtttcttt ggtcggtttt atgtacctat cttcttaagt agctgaagct ccggttttga 4561 actatgcgct cggggttggc gagtgtgttt tgtgaagttt tttaggcacc ttttgaaatg 4621 taatcatttg ggtcaatatg taattttcag tgttagactt gtaaattgtc cgctaaattc 4681 tggccgtttt tggctttttt gttagacaac atgaccgagt acaagcccac ggtgcgcctc 4741 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac 4801 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa 4861 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc 4921 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag 4981 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggag 5041 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc 5101 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcc 5161 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc 5221 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc 5281 tggtgcatga cccgcaagcc cggtgcctag gctagtatgt aagcctagtc ttagataata 5341 aaatcgctat ccatcgaaga tggatgtgtg ttggtttttt gtgtgtgtaa cgctaggcgc 5401 gcctggtgta ccgagaacga tcctctcagt gcgagtctcg acgatccata tcgttgcttg 5461 gcagtcagcc agtcggaatc cagcttggga cccaggaagt ccaatcgtca gatattgtac 5521 tcaagcctgg tcacggcagc gtaccgatct gtttaaacct agatattgat agtctgatcg 5581 gtcaacgtat aatcgagtcc tagcttttgc aaacatctat caagagacag gatcagcagg 5641 aggctttcgc atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 5701 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 5761 gttgggtgcg cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 5821 ttttcgcccc gaagaacgct ttccaatgat gagcactttt aaagttctgc tatgtggcgc 5881 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 5941 gaatgacttg gttgagtatt caccagtcac agaaaagcat cttacggatg gcatgacagt 6001 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 6061 gacaacgatt ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 6121 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 6181 caccacgatg cctgtagcaa tggcaacaac cttgcgtaaa ctattaactg gcgaactact 6241 tactctagct tcccggcaac agttgataga ctggatggag gcggataaag ttgcaggacc 6301 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 6361 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 6421 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 6481 gataggtgcc tcactgatta agcattggta accgattcta ggtgcattgg cgcagaaaaa 6541 aatgcctgat gcgacgctgc gcgtcttata ctcccacata tgccagattc agcaacggat 6601 acggcttccc caacttgccc acttccatac gtgtcctcct taccagaaat ttatccttaa 6661 gatcgtttaa actcgactct ggctctatcg aatctccgtc gtttcgagct tacgcgaaca 6721 gccgtggcgc tcatttgctc gtcgggcatc gaatctcgtc agctatcgtc agcttacctt 6781 tttggcagcg atcgcggctc ccgacatctt ggaccattag ctccacaggt atcttcttcc 6841 ctctagtggt cataacagca gcttcagcta cctctcaatt caaaaaaccc ctcaagaccc 6901 gtttagaggc cccaaggggt tatgctatca atcgttgcgt tacacacaca aaaaaccaac 6961 acacatccat cttcgatgga tagcgatttt attatctaac tgctgatcga gtgtagccag 7021 atctagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca 7081 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca 7141 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg 7201 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg 7261 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc 7321 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatgctg 7381 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca 7441 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt 7501 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg 7561 gaggtctata taagcagagc tggtttagtg aaccgtcaga tcagatcttt gtcgatccta 7621 ccatccactc gacacacccg ccagcggccg c //