LOCUS Exported 7112 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 leader protein. ACCESSION . VERSION . KEYWORDS pGBW-m4133692 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7112) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7112) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7112 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 545..565 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 566..583 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 652..773 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(663..682) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 717..736 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 888..934 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 964..1100 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1145..1216 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1442..2030) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1522..1541) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2530..3739 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3576..3595 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3743..4771 /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 4798..4846 /note="synthetic polyadenylation signal" CDS 5112..5972 /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(5330..5349) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5985..6121 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6266..6337 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6342..6388 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6406..6454 /note="synthetic polyadenylation signal" enhancer 6535..6838 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6840..7042 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6993..7013 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 7111..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatggag tctctcgttc ccggctttaa tgaaaaaact catgtccagt tatccttgcc 61 agtgcttcaa gtaagggatg tgctggtgcg gggtttcgga gacagtgtag aggaagtcct 121 gtcagaggct agacagcacc tgaaggatgg gacctgtggc ttagtggaag ttgagaaggg 181 agtcctcccg cagctggaac aaccttatgt gtttataaaa cgcagcgacg cgcgaacagc 241 acctcacggg catgtaatgg tggagcttgt cgccgagttg gagggcatcc agtacggaag 301 atcgggtgaa acgctgggag ttctagtgcc ccacgttggc gaaattccgg tggcctaccg 361 taaggttttg ctcaggaaaa acggaaataa gggggctggt ggccatagct atggggcaga 421 cctgaagtct ttcgatctgg gcgacgagct gggcaccgac ccatatgagg atttccagga 481 aaactggaac acaaaacaca gctccggggt aactcgcgag ctaatgcgag aattgaatgg 541 aggggaaaac ctttactttc agtcgcacca tcaccatcac cattaaggat ccgcaggcct 601 ctgctagctt gactgactga gatacagcgt accttcagct cacagacatg ataagataca 661 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa 721 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca 781 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca 841 agtaaaacct ctacaaatgt ggtattggcc catctctatc ggtatcgtag cataacccct 901 tggggcctct aaacgggtct tgaggggttt tttgtgcccc tcgggccgga ttgctatcta 961 ccggcattgg cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata ctcccacata 1021 tgccagattc agcaacggat acggcttccc caacttgccc acttccatac gtgtcctcct 1081 taccagaaat ttatccttaa ggtcgtcagc tatcctgcag gcgatctctc gatttcgatc 1141 aagacattcc tttaatggtc ttttctggac accactaggg gtcagaagta gttcatcaaa 1201 ctttcttccc tccctaatct cattggttac cttgggctat cgaaacttaa ttaaccagtc 1261 aagtcagcta cttggcgaga tcgacttgtc tgggtttcga ctacgctcag aattgcgtca 1321 gtcaagttcg atctggtcct tgctattgca cccgttctcc gattacgagt ttcatttaaa 1381 tcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 1441 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 1501 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 1561 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 1621 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 1681 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 1741 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 1801 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 1861 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 1921 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 1981 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 2041 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 2101 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 2161 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 2221 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcattt aaatttccga 2281 actctccaag gccctcgtcg gaaaatcttc aaacctttcg tccgatccat cttgcaggct 2341 acctctcgaa cgaactatcg caagtctctt ggccggcctt gcgccttggc tattgcttgg 2401 cagcgcctat cgccaggtat tactccaatc ccgaatatcc gagatcggga tcacccgaga 2461 gaagttcaac ctacatcctc aatcccgatc tatccgagat ccgaggaata tcgaaatcgg 2521 ggcgcgcctg gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc ctcgtcacgg 2581 cgagcgctgc cacgtcagac gaagggcgca ggagcgtcct gatccttccg cccggacgct 2641 caggacagcg gcccgctgct cataagactc ggccttagaa ccccagtatc agcagaagga 2701 cattttagga cgggacttgg gtgactctag ggcactggtt ttctttccag agagcggaac 2761 aggcgaggaa aagtagtccc ttctcggcga ttctgcggag ggatctccgt ggggcggtga 2821 acgccgatga ttatataagg acgcgccggg tgtggcacag ctagttccgt cgcagccggg 2881 atttgggtcg cggttcttgt ttgtggatcg ctgtgatcgt cacttggtga gtagcgggct 2941 gctgggctgg ccggggcttt cgtggccgcc gggccgctcg gtgggacgga agcgtgtgga 3001 gagaccgcca agggctgtag tctgggtccg cgagcaaggt tgccctgaac tgggggttgg 3061 ggggagcgca gcaaaatggc ggctgttccc gagtcttgaa tggaagacgc ttgtgaggcg 3121 ggctgtgagg tcgttgaaac aaggtggggg gcatggtggg cggcaagaac ccaaggtctt 3181 gagcccttcg ctaatgcggg aaagctctta ttcgggtgag atgggctggg caccatctgg 3241 ggaccctgac gtgaagtttg tcactgactg gagaactcgg tttgtcgtct gttgcggggg 3301 cggcagttat ggcggtgccg ttgggcagtg cacccgtacc tttgggagcg cgcgccctcg 3361 tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca gggtggggcc acctgccggt 3421 aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt tcgggcctag ggtaggctct 3481 cctgaatcga caggcgccgg acctctggtg aggggaggga taagtgaggc gtcagtttct 3541 ttggtcggtt ttatgtacct atcttcttaa gtagctgaag ctccggtttt gaactatgcg 3601 ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca ccttttgaaa tgtaatcatt 3661 tgggtcaata tgtaattttc agtgttagac ttgtaaattg tccgctaaat tctggccgtt 3721 tttggctttt ttgttagaca acatgggtaa aaagcctgaa ctcaccgcga cgtctgtcga 3781 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga 3841 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag 3901 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat ccgccgcgct 3961 cccgattccg gaagtgcttg acattgggga gttcagcgag agcctgacct attgcatctc 4021 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct 4081 gcagccggtc gcggaggcaa tggatgccat cgctgccgcc gatcttagcc agacgagcgg 4141 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg 4201 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc 4261 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg 4321 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac 4381 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat 4441 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag 4501 gcatccggag cttgcaggat cgccccggct ccgggcgtat atgctccgca ttggtcttga 4561 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg 4621 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag 4681 aagcgccgcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg 4741 ccccagcact cgtccgaggg caaaggaata agctagtatg taagcctagt cttagataat 4801 aaaatcgcta tccatcgaag atggatgtgt gttggttttt tgtgtgtgta acgctaggcg 4861 cgcctggtgt accgagaacg atcctctcag tgcgagtctc gacgatccat atcgttgctt 4921 ggcagtcagc cagtcggaat ccagcttggg acccaggaag tccaatcgtc agatattgta 4981 ctcaagcctg gtcacggcag cgtaccgatc tgtttaaacc tagatattga tagtctgatc 5041 ggtcaacgta taatcgagtc ctagcttttg caaacatcta tcaagagaca ggatcagcag 5101 gaggctttcg catgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 5161 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 5221 agttgggtgc gcgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 5281 gttttcgccc cgaagaacgc tttccaatga tgagcacttt taaagttctg ctatgtggcg 5341 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 5401 agaatgactt ggttgagtat tcaccagtca cagaaaagca tcttacggat ggcatgacag 5461 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 5521 tgacaacgat tggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 5581 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 5641 acaccacgat gcctgtagca atggcaacaa ccttgcgtaa actattaact ggcgaactac 5701 ttactctagc ttcccggcaa cagttgatag actggatgga ggcggataaa gttgcaggac 5761 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 5821 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 5881 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 5941 agataggtgc ctcactgatt aagcattggt aaccgattct aggtgcattg gcgcagaaaa 6001 aaatgcctga tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga 6061 tacggcttcc ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta 6121 agatcgttta aactcgactc tggctctatc gaatctccgt cgtttcgagc ttacgcgaac 6181 agccgtggcg ctcatttgct cgtcgggcat cgaatctcgt cagctatcgt cagcttacct 6241 ttttggcagc gatcgcggct cccgacatct tggaccatta gctccacagg tatcttcttc 6301 cctctagtgg tcataacagc agcttcagct acctctcaat tcaaaaaacc cctcaagacc 6361 cgtttagagg ccccaagggg ttatgctatc aatcgttgcg ttacacacac aaaaaaccaa 6421 cacacatcca tcttcgatgg atagcgattt tattatctaa ctgctgatcg agtgtagcca 6481 gatctagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac 6541 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc 6601 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt 6661 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 6721 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 6781 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct 6841 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 6901 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 6961 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 7021 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atcagatctt tgtcgatcct 7081 accatccact cgacacaccc gccagcggcc gc //