LOCUS Exported 7010 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression plasmid for SARS-CoV-2 ORF3a protein. ACCESSION . VERSION . KEYWORDS pGBW-m4134461 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7010) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 7010) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7010 /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 1408..1428 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1432..1497 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 1550..1671 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1561..1580) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1615..1634 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 1786..1832 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 1862..1998 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 2043..2114 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(2340..2928) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2420..2439) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 3428..4637 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4474..4493 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 4641..5240 /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(4641..4660) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5137..5157 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 5267..5315 /note="synthetic polyadenylation signal" CDS 5581..6441 /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(5799..5818) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 6454..6590 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6742..6813 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 6818..6864 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 6882..6930 /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 ccatggatct gtttatgcgt 601 atattcacca tcggcacggt tacattgaag caaggggaaa ttaaagacgc aactcccagt 661 gacttcgtac gggcgaccgc tacaattccg atccaggcca gcctcccttt tggatggctt 721 atcgtcggcg tggccctact ggctgtgttt cagtcggcat caaagataat cactctgaaa 781 aagcgatggc agttagccct tagcaaaggt gtgcacttcg tatgcaattt gctgctcctg 841 ttcgtcaccg tttattccca tctgctcctg gtggctgccg gattggaggc accattttta 901 tacctatacg ccctcgtgta ttttctgcaa tctattaact tcgtccgcat tatcatgaga 961 ctctggctct gttggaagtg caggtctaaa aatcccttgt tatatgatgc caactacttc 1021 ctgtgttggc acactaactg ctatgactac tgtattccat acaattcagt tacctccagt 1081 atagtgatca catctgggga tggtacgact tcgcctattt ccgagcatga ctatcagatc 1141 ggcgggtaca cagaaaagtg ggagagcgga gtcaaggatt gcgtggtgct tcactcctat 1201 ttcaccagtg actactacca gctttatagc acacaacttt caacagacac cggcgtggag 1261 catgtgactt ttttcattta taataaaatc gttgatgaac cggaggaaca cgttcagata 1321 cacactatcg acggcagctc tggtgtcgtc aaccctgtta tggagcccat ttacgatgaa 1381 ccaacgacca caacatctgt acctctggaa aacctttact ttcagtcgat ggattataag 1441 gaccacgatg gagactacaa agatcatgac attgattaca aggacgacga tgacaaataa 1501 gctagcttga ctgactgaga tacagcgtac cttcagctca cagacatgat aagatacatt 1561 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt 1621 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac 1681 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag 1741 taaaacctct acaaatgtgg tattggccca tctctatcgg tatcgtagca taaccccttg 1801 gggcctctaa acgggtcttg aggggttttt tgtgcccctc gggccggatt gctatctacc 1861 ggcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact cccacatatg 1921 ccagattcag caacggatac ggcttcccca acttgcccac ttccatacgt gtcctcctta 1981 ccagaaattt atccttaagg tcgtcagcta tcctgcaggc gatctctcga tttcgatcaa 2041 gacattcctt taatggtctt ttctggacac cactaggggt cagaagtagt tcatcaaact 2101 ttcttccctc cctaatctca ttggttacct tgggctatcg aaacttaatt aaccagtcaa 2161 gtcagctact tggcgagatc gacttgtctg ggtttcgact acgctcagaa ttgcgtcagt 2221 caagttcgat ctggtccttg ctattgcacc cgttctccga ttacgagttt catttaaatc 2281 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 2341 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 2401 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 2461 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 2521 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 2581 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 2641 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 2701 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 2761 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 2821 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 2881 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 2941 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3001 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 3061 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 3121 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcatttaa atttccgaac 3181 tctccaaggc cctcgtcgga aaatcttcaa acctttcgtc cgatccatct tgcaggctac 3241 ctctcgaacg aactatcgca agtctcttgg ccggccttgc gccttggcta ttgcttggca 3301 gcgcctatcg ccaggtatta ctccaatccc gaatatccga gatcgggatc acccgagaga 3361 agttcaacct acatcctcaa tcccgatcta tccgagatcc gaggaatatc gaaatcgggg 3421 cgcgcctggc ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct cgtcacggcg 3481 agcgctgcca cgtcagacga agggcgcagg agcgtcctga tccttccgcc cggacgctca 3541 ggacagcggc ccgctgctca taagactcgg ccttagaacc ccagtatcag cagaaggaca 3601 ttttaggacg ggacttgggt gactctaggg cactggtttt ctttccagag agcggaacag 3661 gcgaggaaaa gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac 3721 gccgatgatt atataaggac gcgccgggtg tggcacagct agttccgtcg cagccgggat 3781 ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtca cttggtgagt agcgggctgc 3841 tgggctggcc ggggctttcg tggccgccgg gccgctcggt gggacggaag cgtgtggaga 3901 gaccgccaag ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg ggggttgggg 3961 ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg 4021 ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc aaggtcttga 4081 gcccttcgct aatgcgggaa agctcttatt cgggtgagat gggctgggca ccatctgggg 4141 accctgacgt gaagtttgtc actgactgga gaactcggtt tgtcgtctgt tgcgggggcg 4201 gcagttatgg cggtgccgtt gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc 4261 gtgtcgtgac gtcacccgtt ctgttggctt ataatgcagg gtggggccac ctgccggtag 4321 gtgtgcggta ggcttttctc cgtcgcagga cgcagggttc gggcctaggg taggctctcc 4381 tgaatcgaca ggcgccggac ctctggtgag gggagggata agtgaggcgt cagtttcttt 4441 ggtcggtttt atgtacctat cttcttaagt agctgaagct ccggttttga actatgcgct 4501 cggggttggc gagtgtgttt tgtgaagttt tttaggcacc ttttgaaatg taatcatttg 4561 ggtcaatatg taattttcag tgttagactt gtaaattgtc cgctaaattc tggccgtttt 4621 tggctttttt gttagacaac atgaccgagt acaagcccac ggtgcgcctc gccacccgcg 4681 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc 4741 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc 4801 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg 4861 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc 4921 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggag ggcctcctgg 4981 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc 5041 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcc gccgagcgcg 5101 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc 5161 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga 5221 cccgcaagcc cggtgcctag gctagtatgt aagcctagtc ttagataata aaatcgctat 5281 ccatcgaaga tggatgtgtg ttggtttttt gtgtgtgtaa cgctaggcgc gcctggtgta 5341 ccgagaacga tcctctcagt gcgagtctcg acgatccata tcgttgcttg gcagtcagcc 5401 agtcggaatc cagcttggga cccaggaagt ccaatcgtca gatattgtac tcaagcctgg 5461 tcacggcagc gtaccgatct gtttaaacct agatattgat agtctgatcg gtcaacgtat 5521 aatcgagtcc tagcttttgc aaacatctat caagagacag gatcagcagg aggctttcgc 5581 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 5641 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgcg 5701 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 5761 gaagaacgct ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 5821 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 5881 gttgagtatt caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 5941 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatt 6001 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 6061 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 6121 cctgtagcaa tggcaacaac cttgcgtaaa ctattaactg gcgaactact tactctagct 6181 tcccggcaac agttgataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 6241 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 6301 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 6361 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 6421 tcactgatta agcattggta accgattcta ggtgcattgg cgcagaaaaa aatgcctgat 6481 gcgacgctgc gcgtcttata ctcccacata tgccagattc agcaacggat acggcttccc 6541 caacttgccc acttccatac gtgtcctcct taccagaaat ttatccttaa gatcccgaat 6601 cgtttaaact cgactctggc tctatcgaat ctccgtcgtt tcgagcttac gcgaacagcc 6661 gtggcgctca tttgctcgtc gggcatcgaa tctcgtcagc tatcgtcagc ttaccttttt 6721 ggcagcgatc gcggctcccg acatcttgga ccattagctc cacaggtatc ttcttccctc 6781 tagtggtcat aacagcagct tcagctacct ctcaattcaa aaaacccctc aagacccgtt 6841 tagaggcccc aaggggttat gctatcaatc gttgcgttac acacacaaaa aaccaacaca 6901 catccatctt cgatggatag cgattttatt atctaactgc tgatcgagtg tagccagatc 6961 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg //