LOCUS Exported 6299 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 ORF10 protein. ACCESSION . VERSION . KEYWORDS pGBW-m4252818 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6299) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6299) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6299 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 119..139 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 143..208 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 261..382 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(272..291) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 326..345 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 497..543 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 573..709 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 754..825 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1051..1639) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1131..1150) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2139..3348 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3185..3204 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3352..3951 /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(3352..3371) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3848..3868 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 3978..4026 /note="synthetic polyadenylation signal" CDS 4292..5152 /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(4510..4529) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5165..5301 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 5453..5524 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 5529..5575 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 5593..5641 /note="synthetic polyadenylation signal" enhancer 5722..6025 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6027..6229 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6180..6200 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 6298..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatgggg tacataaatg ttttcgcctt tccattcacg atctattcac tccttttatg 61 taggatgaac tctcgcaatt acattgctca ggtagacgtc gtgaacttta acctaactga 121 aaacctttac tttcagtcga tggattataa ggaccacgat ggagactaca aagatcatga 181 cattgattac aaggacgacg atgacaaata agctagcttg actgactgag atacagcgta 241 ccttcagctc acagacatga taagatacat tgatgagttt ggacaaacca caactagaat 301 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 361 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca 421 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtattggccc 481 atctctatcg gtatcgtagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 541 ttgtgcccct cgggccggat tgctatctac cggcattggc gcagaaaaaa atgcctgatg 601 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc 661 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag gtcgtcagct 721 atcctgcagg cgatctctcg atttcgatca agacattcct ttaatggtct tttctggaca 781 ccactagggg tcagaagtag ttcatcaaac tttcttccct ccctaatctc attggttacc 841 ttgggctatc gaaacttaat taaccagtca agtcagctac ttggcgagat cgacttgtct 901 gggtttcgac tacgctcaga attgcgtcag tcaagttcga tctggtcctt gctattgcac 961 ccgttctccg attacgagtt tcatttaaat catgtgagca aaaggccagc aaaaggccag 1021 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 1081 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 1141 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 1201 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 1261 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 1321 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 1381 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 1441 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 1501 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 1561 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 1621 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 1681 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 1741 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 1801 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 1861 ttcatccata gttgcattta aatttccgaa ctctccaagg ccctcgtcgg aaaatcttca 1921 aacctttcgt ccgatccatc ttgcaggcta cctctcgaac gaactatcgc aagtctcttg 1981 gccggccttg cgccttggct attgcttggc agcgcctatc gccaggtatt actccaatcc 2041 cgaatatccg agatcgggat cacccgagag aagttcaacc tacatcctca atcccgatct 2101 atccgagatc cgaggaatat cgaaatcggg gcgcgcctgg cctccgcgcc gggttttggc 2161 gcctcccgcg ggcgcccccc tcgtcacggc gagcgctgcc acgtcagacg aagggcgcag 2221 gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg 2281 gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg 2341 gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat 2401 tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt 2461 gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc 2521 tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg 2581 ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc 2641 gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg 2701 agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg 2761 catggtgggc ggcaagaacc caaggtcttg agcccttcgc taatgcggga aagctcttat 2821 tcgggtgaga tgggctgggc accatctggg gaccctgacg tgaagtttgt cactgactgg 2881 agaactcggt ttgtcgtctg ttgcgggggc ggcagttatg gcggtgccgt tgggcagtgc 2941 acccgtacct ttgggagcgc gcgccctcgt cgtgtcgtga cgtcacccgt tctgttggct 3001 tataatgcag ggtggggcca ccagccggta ggtgtgcggt aggcttttct ccgtcgcagg 3061 acgcagggtt cgggcctagg gtaggctctc ctgaatcgac aggcgccgga cctctggtga 3121 ggggagggat aagtgaggcg tcagtttctt tggtcggttt tatgtaccta tcttcttaag 3181 tagctgaagc tccggttttg aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt 3241 ttttaggcac cttttgaaat gtaatcattt gggtcaatat gtaattttca gtgttagact 3301 tgtaaattgt ccgctaaatt ctggccgttt ttggcttttt tgttagacaa catgaccgag 3361 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc 3421 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc 3481 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag 3541 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa 3601 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg 3661 gccgcgcagc aacagatgga gggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg 3721 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc 3781 gtgctccccg gagtggaggc cgccgagcgc gccggggtgc ccgccttcct ggagacctcc 3841 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag 3901 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccta ggctagtatg 3961 taagcctagt cttagataat aaaatcgcta tccatcgaag atggatgtgt gttggttttt 4021 tgtgtgtgta acgctaggcg cgcctggtgt accgagaacg atcctctcag tgcgagtctc 4081 gacgatccat atcgttgctt ggcagtcagc cagtcggaat ccagcttggg acccaggaag 4141 tccaatcgtc agatattgta ctcaagcctg gtcacggcag cgtaccgatc tgtttaaacc 4201 tagatattga tagtctgatc ggtcaacgta taatcgagtc ctagcttttg caaacatcta 4261 tcaagagaca ggatcagcag gaggctttcg catgagtatt caacatttcc gtgtcgccct 4321 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 4381 agtaaaagat gctgaagatc agttgggtgc gcgagtgggt tacatcgaac tggatctcaa 4441 cagcggtaag atccttgaga gttttcgccc cgaagaacgc tttccaatga tgagcacttt 4501 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 4561 tcgccgcata cactattctc agaatgactt ggttgagtat tcaccagtca cagaaaagca 4621 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 4681 cactgcggcc aacttacttc tgacaacgat tggaggaccg aaggagctaa ccgctttttt 4741 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 4801 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa ccttgcgtaa 4861 actattaact ggcgaactac ttactctagc ttcccggcaa cagttgatag actggatgga 4921 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 4981 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 5041 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 5101 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aaccgattct 5161 aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat 5221 atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc 5281 ttaccagaaa tttatcctta agatcccgaa tcgtttaaac tcgactctgg ctctatcgaa 5341 tctccgtcgt ttcgagctta cgcgaacagc cgtggcgctc atttgctcgt cgggcatcga 5401 atctcgtcag ctatcgtcag cttacctttt tggcagcgat cgcggctccc gacatcttgg 5461 accattagct ccacaggtat cttcttccct ctagtggtca taacagcagc ttcagctacc 5521 tctcaattca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctatcaat 5581 cgttgcgtta cacacacaaa aaaccaacac acatccatct tcgatggata gcgattttat 5641 tatctaactg ctgatcgagt gtagccagat ctagtaatca attacggggt cattagttca 5701 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 5761 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 5821 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 5881 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 5941 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 6001 cgtattagtc atcgctatta ccatgctgat gcggttttgg cagtacatca atgggcgtgg 6061 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 6121 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 6181 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa 6241 ccgtcagatc agatctttgt cgatcctacc atccactcga cacacccgcc agcggccgc //