LOCUS Exported 6554 bp ds-DNA circular SYN 09-DEC-2022 DEFINITION Mammalian expression plasmid for SARS-CoV-2 nsp10. ACCESSION . VERSION . KEYWORDS pGBW-m4134309 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6554) TITLE SARS-CoV-2 protein Mammalian expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 6554) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 9, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6554 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 425..445 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 446..463 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" polyA_signal 516..637 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(527..546) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 581..600 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 752..798 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 828..964 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 1009..1080 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(1306..1894) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1386..1405) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 2394..3603 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3440..3459 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 3607..4206 /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(3607..3626) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4103..4123 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 4233..4281 /note="synthetic polyadenylation signal" CDS 4547..5407 /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(4765..4784) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" terminator 5420..5556 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 5708..5779 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 5784..5830 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 5848..5896 /note="synthetic polyadenylation signal" enhancer 5977..6280 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6282..6484 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6435..6455 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 6553..8 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 caccatggca gggaatgcga cagaggttcc tgccaactca acggtcttgt ctttttgtgc 61 tttcgccgtg gacgctgcaa aagcctacaa ggattatcta gcctcgggtg gacagccaat 121 cactaactgc gtaaagatgc tttgtaccca caccggcact ggccaagcaa ttacagtgac 181 ccccgaagct aatatggacc aggagtcctt cggaggtgcg agctgctgcc tgtactgtcg 241 ttgtcatata gatcacccga atcccaaagg gttttgcgac ctcaagggca aatatgtcca 301 gatcccaacg acatgcgcca acgatcctgt gggattcact ctgaagaaca ccgtatgtac 361 cgtttgcggg atgtggaagg ggtatggctg tagttgcgac caattacggg aaccgatgct 421 gcaagaaaac ctttactttc agtcgcacca tcaccatcac cattaagcta gcttgactga 481 ctgagataca gcgtaccttc agctcacaga catgataaga tacattgatg agtttggaca 541 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc 601 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt 661 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa 721 atgtggtatt ggcccatctc tatcggtatc gtagcataac cccttggggc ctctaaacgg 781 gtcttgaggg gttttttgtg cccctcgggc cggattgcta tctaccggca ttggcgcaga 841 aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac 901 ggatacggct tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc 961 ttaaggtcgt cagctatcct gcaggcgatc tctcgatttc gatcaagaca ttcctttaat 1021 ggtcttttct ggacaccact aggggtcaga agtagttcat caaactttct tccctcccta 1081 atctcattgg ttaccttggg ctatcgaaac ttaattaacc agtcaagtca gctacttggc 1141 gagatcgact tgtctgggtt tcgactacgc tcagaattgc gtcagtcaag ttcgatctgg 1201 tccttgctat tgcacccgtt ctccgattac gagtttcatt taaatcatgt gagcaaaagg 1261 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 1321 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 1381 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 1441 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 1501 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 1561 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 1621 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 1681 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 1741 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 1801 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 1861 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 1921 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 1981 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 2041 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 2101 gatctgtcta tttcgttcat ccatagttgc atttaaattt ccgaactctc caaggccctc 2161 gtcggaaaat cttcaaacct ttcgtccgat ccatcttgca ggctacctct cgaacgaact 2221 atcgcaagtc tcttggccgg ccttgcgcct tggctattgc ttggcagcgc ctatcgccag 2281 gtattactcc aatcccgaat atccgagatc gggatcaccc gagagaagtt caacctacat 2341 cctcaatccc gatctatccg agatccgagg aatatcgaaa tcggggcgcg cctggcctcc 2401 gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcgtc acggcgagcg ctgccacgtc 2461 agacgaaggg cgcaggagcg tcctgatcct tccgcccgga cgctcaggac agcggcccgc 2521 tgctcataag actcggcctt agaaccccag tatcagcaga aggacatttt aggacgggac 2581 ttgggtgact ctagggcact ggttttcttt ccagagagcg gaacaggcga ggaaaagtag 2641 tcccttctcg gcgattctgc ggagggatct ccgtggggcg gtgaacgccg atgattatat 2701 aaggacgcgc cgggtgtggc acagctagtt ccgtcgcagc cgggatttgg gtcgcggttc 2761 ttgtttgtgg atcgctgtga tcgtcacttg gtgagtagcg ggctgctggg ctggccgggg 2821 ctttcgtggc cgccgggccg ctcggtggga cggaagcgtg tggagagacc gccaagggct 2881 gtagtctggg tccgcgagca aggttgccct gaactggggg ttggggggag cgcagcaaaa 2941 tggcggctgt tcccgagtct tgaatggaag acgcttgtga ggcgggctgt gaggtcgttg 3001 aaacaaggtg gggggcatgg tgggcggcaa gaacccaagg tcttgagccc ttcgctaatg 3061 cgggaaagct cttattcggg tgagatgggc tgggcaccat ctggggaccc tgacgtgaag 3121 tttgtcactg actggagaac tcggtttgtc gtctgttgcg ggggcggcag ttatggcggt 3181 gccgttgggc agtgcacccg tacctttggg agcgcgcgcc ctcgtcgtgt cgtgacgtca 3241 cccgttctgt tggcttataa tgcagggtgg ggccacctgc cggtaggtgt gcggtaggct 3301 tttctccgtc gcaggacgca gggttcgggc ctagggtagg ctctcctgaa tcgacaggcg 3361 ccggacctct ggtgagggga gggataagtg aggcgtcagt ttctttggtc ggttttatgt 3421 acctatcttc ttaagtagct gaagctccgg ttttgaacta tgcgctcggg gttggcgagt 3481 gtgttttgtg aagtttttta ggcacctttt gaaatgtaat catttgggtc aatatgtaat 3541 tttcagtgtt agacttgtaa attgtccgct aaattctggc cgtttttggc ttttttgtta 3601 gacaacatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg 3661 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat 3721 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg 3781 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg 3841 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg 3901 agcggttccc ggctggccgc gcagcaacag atggagggcc tcctggcgcc gcaccggccc 3961 aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt 4021 ctgggcagcg ccgtcgtgct ccccggagtg gaggccgccg agcgcgccgg ggtgcccgcc 4081 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc 4141 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt 4201 gcctaggcta gtatgtaagc ctagtcttag ataataaaat cgctatccat cgaagatgga 4261 tgtgtgttgg ttttttgtgt gtgtaacgct aggcgcgcct ggtgtaccga gaacgatcct 4321 ctcagtgcga gtctcgacga tccatatcgt tgcttggcag tcagccagtc ggaatccagc 4381 ttgggaccca ggaagtccaa tcgtcagata ttgtactcaa gcctggtcac ggcagcgtac 4441 cgatctgttt aaacctagat attgatagtc tgatcggtca acgtataatc gagtcctagc 4501 ttttgcaaac atctatcaag agacaggatc agcaggaggc tttcgcatga gtattcaaca 4561 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 4621 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcgcgag tgggttacat 4681 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgctttcc 4741 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 4801 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtattcacc 4861 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 4921 aaccatgagt gataacactg cggccaactt acttctgaca acgattggag gaccgaagga 4981 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 5041 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 5101 aacaaccttg cgtaaactat taactggcga actacttact ctagcttccc ggcaacagtt 5161 gatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 5221 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 5281 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 5341 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 5401 ttggtaaccg attctaggtg cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt 5461 cttatactcc cacatatgcc agattcagca acggatacgg cttccccaac ttgcccactt 5521 ccatacgtgt cctccttacc agaaatttat ccttaagatc ccgaatcgtt taaactcgac 5581 tctggctcta tcgaatctcc gtcgtttcga gcttacgcga acagccgtgg cgctcatttg 5641 ctcgtcgggc atcgaatctc gtcagctatc gtcagcttac ctttttggca gcgatcgcgg 5701 ctcccgacat cttggaccat tagctccaca ggtatcttct tccctctagt ggtcataaca 5761 gcagcttcag ctacctctca attcaaaaaa cccctcaaga cccgtttaga ggccccaagg 5821 ggttatgcta tcaatcgttg cgttacacac acaaaaaacc aacacacatc catcttcgat 5881 ggatagcgat tttattatct aactgctgat cgagtgtagc cagatctagt aatcaattac 5941 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 6001 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 6061 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 6121 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 6181 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 6241 ttggcagtac atctacgtat tagtcatcgc tattaccatg ctgatgcggt tttggcagta 6301 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 6361 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 6421 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 6481 agctggttta gtgaaccgtc agatcagatc tttgtcgatc ctaccatcca ctcgacacac 6541 ccgccagcgg ccgc //