LOCUS Exported 6725 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 leader protein. ACCESSION . VERSION . KEYWORDS pGBW-m4133843 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6725) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6725) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6725 /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 1123..1143 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1147..1212 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 1265..1386 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1276..1295) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1330..1349 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1501..1547 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1577..1713 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1758..1829 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2055..2643) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2135..2154) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3143..4352 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4189..4208 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4356..4955 /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(4356..4375) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4852..4872 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4982..5030 /note="synthetic polyadenylation signal" CDS 5296..6156 /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(5514..5533) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6169..6305 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6457..6528 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6533..6579 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6597..6645 /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 ccatggagtc tctcgttccc 601 ggctttaatg aaaaaactca tgtccagtta tccttgccag tgcttcaagt aagggatgtg 661 ctggtgcggg gtttcggaga cagtgtagag gaagtcctgt cagaggctag acagcacctg 721 aaggatggga cctgtggctt agtggaagtt gagaagggag tcctcccgca gctggaacaa 781 ccttatgtgt ttataaaacg cagcgacgcg cgaacagcac ctcacgggca tgtaatggtg 841 gagcttgtcg ccgagttgga gggcatccag tacggaagat cgggtgaaac gctgggagtt 901 ctagtgcccc acgttggcga aattccggtg gcctaccgta aggttttgct caggaaaaac 961 ggaaataagg gggctggtgg ccatagctat ggggcagacc tgaagtcttt cgatctgggc 1021 gacgagctgg gcaccgaccc atatgaggat ttccaggaaa actggaacac aaaacacagc 1081 tccggggtaa ctcgcgagct aatgcgagaa ttgaatggag gggaaaacct ttactttcag 1141 tcgatggatt ataaggacca cgatggagac tacaaagatc atgacattga ttacaaggac 1201 gacgatgaca aataagctag cttgactgac tgagatacag cgtaccttca gctcacagac 1261 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 1321 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 1381 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag 1441 gttttttaaa gcaagtaaaa cctctacaaa tgtggtattg gcccatctct atcggtatcg 1501 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgtgc ccctcgggcc 1561 ggattgctat ctaccggcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt 1621 atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca 1681 tacgtgtcct ccttaccaga aatttatcct taaggtcgtc agctatcctg caggcgatct 1741 ctcgatttcg atcaagacat tcctttaatg gtcttttctg gacaccacta ggggtcagaa 1801 gtagttcatc aaactttctt ccctccctaa tctcattggt taccttgggc tatcgaaact 1861 taattaacca gtcaagtcag ctacttggcg agatcgactt gtctgggttt cgactacgct 1921 cagaattgcg tcagtcaagt tcgatctggt ccttgctatt gcacccgttc tccgattacg 1981 agtttcattt aaatcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 2041 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 2101 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 2161 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 2221 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 2281 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 2341 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 2401 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 2461 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 2521 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 2581 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 2641 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 2701 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 2761 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 2821 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgca 2881 tttaaatttc cgaactctcc aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc 2941 catcttgcag gctacctctc gaacgaacta tcgcaagtct cttggccggc cttgcgcctt 3001 ggctattgct tggcagcgcc tatcgccagg tattactcca atcccgaata tccgagatcg 3061 ggatcacccg agagaagttc aacctacatc ctcaatcccg atctatccga gatccgagga 3121 atatcgaaat cggggcgcgc ctggcctccg cgccgggttt tggcgcctcc cgcgggcgcc 3181 cccctcgtca cggcgagcgc tgccacgtca gacgaagggc gcaggagcgt cctgatcctt 3241 ccgcccggac gctcaggaca gcggcccgct gctcataaga ctcggcctta gaaccccagt 3301 atcagcagaa ggacatttta ggacgggact tgggtgactc tagggcactg gttttctttc 3361 cagagagcgg aacaggcgag gaaaagtagt cccttctcgg cgattctgcg gagggatctc 3421 cgtggggcgg tgaacgccga tgattatata aggacgcgcc gggtgtggca cagctagttc 3481 cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg 3541 tgagtagcgg gctgctgggc tggccggggc tttcgtggcc gccgggccgc tcggtgggac 3601 ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg 3661 aactgggggt tggggggagc gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga 3721 cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt gggcggcaag 3781 aacccaaggt cttgagccct tcgctaatgc gggaaagctc ttattcgggt gagatgggct 3841 gggcaccatc tggggaccct gacgtgaagt ttgtcactga ctggagaact cggtttgtcg 3901 tctgttgcgg gggcggcagt tatggcggtg ccgttgggca gtgcacccgt acctttggga 3961 gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt ggcttataat gcagggtggg 4021 gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg caggacgcag ggttcgggcc 4081 tagggtaggc tctcctgaat cgacaggcgc cggacctctg gtgaggggag ggataagtga 4141 ggcgtcagtt tctttggtcg gttttatgta cctatcttct taagtagctg aagctccggt 4201 tttgaactat gcgctcgggg ttggcgagtg tgttttgtga agttttttag gcaccttttg 4261 aaatgtaatc atttgggtca atatgtaatt ttcagtgtta gacttgtaaa ttgtccgcta 4321 aattctggcc gtttttggct tttttgttag acaacatgac cgagtacaag cccacggtgc 4381 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg 4441 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc 4501 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg 4561 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg 4621 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga 4681 tggagggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg 4741 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg 4801 aggccgccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc 4861 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc 4921 gcacctggtg catgacccgc aagcccggtg cctaggctag tatgtaagcc tagtcttaga 4981 taataaaatc gctatccatc gaagatggat gtgtgttggt tttttgtgtg tgtaacgcta 5041 ggcgcgcctg gtgtaccgag aacgatcctc tcagtgcgag tctcgacgat ccatatcgtt 5101 gcttggcagt cagccagtcg gaatccagct tgggacccag gaagtccaat cgtcagatat 5161 tgtactcaag cctggtcacg gcagcgtacc gatctgttta aacctagata ttgatagtct 5221 gatcggtcaa cgtataatcg agtcctagct tttgcaaaca tctatcaaga gacaggatca 5281 gcaggaggct ttcgcatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 5341 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 5401 gatcagttgg gtgcgcgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 5461 gagagttttc gccccgaaga acgctttcca atgatgagca cttttaaagt tctgctatgt 5521 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 5581 tctcagaatg acttggttga gtattcacca gtcacagaaa agcatcttac ggatggcatg 5641 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 5701 cttctgacaa cgattggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 5761 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 5821 cgtgacacca cgatgcctgt agcaatggca acaaccttgc gtaaactatt aactggcgaa 5881 ctacttactc tagcttcccg gcaacagttg atagactgga tggaggcgga taaagttgca 5941 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 6001 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 6061 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 6121 gctgagatag gtgcctcact gattaagcat tggtaaccga ttctaggtgc attggcgcag 6181 aaaaaaatgc ctgatgcgac gctgcgcgtc ttatactccc acatatgcca gattcagcaa 6241 cggatacggc ttccccaact tgcccacttc catacgtgtc ctccttacca gaaatttatc 6301 cttaagatcc cgaatcgttt aaactcgact ctggctctat cgaatctccg tcgtttcgag 6361 cttacgcgaa cagccgtggc gctcatttgc tcgtcgggca tcgaatctcg tcagctatcg 6421 tcagcttacc tttttggcag cgatcgcggc tcccgacatc ttggaccatt agctccacag 6481 gtatcttctt ccctctagtg gtcataacag cagcttcagc tacctctcaa ttcaaaaaac 6541 ccctcaagac ccgtttagag gccccaaggg gttatgctat caatcgttgc gttacacaca 6601 caaaaaacca acacacatcc atcttcgatg gatagcgatt ttattatcta actgctgatc 6661 gagtgtagcc agatctagta atcaattacg gggtcattag ttcatagccc atatatggag 6721 ttccg //