LOCUS Exported 9567 bp ds-DNA circular SYN 03-JUN-2021 DEFINITION Baculoviral transfer vector to co-express SARS-CoV-2 nsp14 and nsp10 in insect cells. ACCESSION . VERSION . KEYWORDS pBIG2ab_nsp14/nsp10-6His-3xFlag (SARS-CoV-2) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9567) AUTHORS Canal B, McClure AW, Curran JF, Wu M, Ulferts R, Weissmann F, Zeng J, Bertolin AP, Milligan JC, Basu S, Drury LS, Deegan T, Fujisawa R, Roberts EL, Basier C, Labib K, Beale R, Howell M, Diffley JFX TITLE Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp14/nsp10 Exoribonuclease JOURNAL bioRxiv https://www.biorxiv.org/content/10.1101/2021.04.07.438812v1 REFERENCE 2 (bases 1 to 9567) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 3, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9567 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 104..195 /gene="polh from Autographa californica" /label=polyhedrin promoter /note="promoter for the baculovirus polyhedrin gene" regulatory 239..248 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 674..691 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 698..763 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 896..1030 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" mobile_element 1346..1511 /mobile_element_type="transposon:Tn7" /note="mini-Tn7 element (left end of the Tn7 transposon)" promoter 1896..1998 /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" CDS 1999..2658 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(2065..2084) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" rep_origin 2958..3412 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3045..3064) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3254..3275 /label=F1ori-F /note="F1 origin, forward primer" promoter 3438..3542 /gene="bla" /label=AmpR promoter CDS 3543..4403 /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(3761..3780) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4574..5162 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5063..5082 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5316..5333 /label=L4440 /note="L4440 vector, forward primer" mobile_element 5467..5691 /mobile_element_type="transposon:Tn7" /note="mini-Tn7 element (right end of the Tn7 transposon)" CDS complement(5758..6291) /codon_start=1 /gene="aacC1" /product="gentamycin acetyltransferase" /label=GmR /note="confers resistance to gentamycin" /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR EEVMHFDIDPSTAT" promoter complement(6480..6508) /gene="intI1 (promoter lies within the coding sequence)" /label=Pc promoter /note="class 1 integron promoter" protein_bind 6904..6937 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." polyA_signal 7035..7083 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" promoter 7178..7269 /gene="polh from Autographa californica" /label=polyhedrin promoter /note="promoter for the baculovirus polyhedrin gene" regulatory 7313..7322 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 9035..9169 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 9528..9 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" ORIGIN 1 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcaag tgaacgctct 61 atggtctaaa gatttaaatc gacctactcc ggaatattaa tagatcatgg agataattaa 121 aatgataacc atctcgcaaa taaataagta ttttactgtt ttcgtaacag ttttgtaata 181 aaaaaaccta taaatattcc ggattattca taccgtccca ccatcgggcg cggatccagc 241 caccatggct ggaaacgcta ccgaggtgcc cgctaactct accgtgctgt ccttctgcgc 301 tttcgctgtg gacgctgcta aggcttacaa ggactacctg gcttccggtg gccagcctat 361 caccaactgc gtgaagatgc tgtgcaccca caccggtact ggacaggcta tcactgtgac 421 ccctgaggct aacatggacc aagagtcctt cggtggtgct tcctgctgcc tgtactgccg 481 ttgccacatc gatcacccca atcctaaggg cttctgcgac ctgaagggca aatacgtgca 541 gatccccacc acttgcgcta acgaccctgt gggtttcacc ctgaagaaca ccgtgtgcac 601 cgtctgcggc atgtggaaag gatacggttg ctcctgcgac cagctgcgcg aacccatgtt 661 gcagggtggc tcccaccatc accaccatca cggttctgac tacaaggacc acgacggcga 721 ttacaaggat catgacatcg actacaaaga cgacgatgac aagtaaaagc ttgtcgagaa 781 gtactagagg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa 841 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt 901 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 961 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 1021 tgtctggatc tgatcactgc ttgagcctag aagatccggc tgctaacaaa gcccgaaagg 1081 aagctgagtt ggctgctgcc accgctgagc aataactatc ataaccattt aaataaatct 1141 atatctcaat cggggttacg tcgatacccc accgagaccc cattgggacc aatacgcccg 1201 cgtttcttcc ttttccccac cccaaccccc aagttcgggt gaaggcccag ggctcgcagc 1261 caacgtcggg gcggcaagcc ctgccatagc cactacgggt taattaaaaa ctggatacta 1321 ttgcacgttt caatcaggag atccgaacca gataagtgaa atctagttcc aaactatttt 1381 gtcattttta attttcgtat tagcttacga cgctacaccc agttcccatc tattttgtca 1441 ctcttcccta aataatcctt aaaaactcca tttccacccc tcccagttcc caactatttt 1501 gtccgcccac agcggggcat ttttcttcct gttatgtttt taatcaaaca tcctgccaac 1561 tccatgtgac aaaccgtcat cttcggctac tttttctctg tcacagaatg aaaatttttc 1621 tgtcatctct tcgttattaa tgtttgtaat tgactgaata tcaacgctta tttgcagcct 1681 gaactgaaca ggagggacag ctgatagaaa cagaagccac tggagcacct caaaaacacc 1741 atcatacact aaatcagtaa gttggcagca tcacccgacg cactttgcgc cgaataaata 1801 cctgtgacgg aagatcactt cgcagaataa ataaatcctg gtgtccctgt tgataccggg 1861 aagccctggg ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag aggttccaac 1921 tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca 1981 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc 2041 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac 2101 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag 2161 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt 2221 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt 2281 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg 2341 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc 2401 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc 2461 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc 2521 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc 2581 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag 2641 tggcagggcg gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggttgc 2701 tacgcctgaa taagtgataa taagcggatg aatggcagaa attcgaaagc aaattcgacc 2761 cggtcgtcgg ttcagggcag ggtcgttaaa tagccgctta tgtctattgc tggtttaccg 2821 gtttattgac taccggaagc agtgtgaccg tgtgcttctc aaatgcctga ggccagtttg 2881 ctcaggctct ccccgtggag gtaataattg acgatatgat catttattct gcctcccaga 2941 gcctgatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 3001 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 3061 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 3121 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 3181 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 3241 cacgttctta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 3301 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 3361 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca 3421 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 3481 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 3541 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 3601 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 3661 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 3721 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 3781 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 3841 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 3901 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 3961 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 4021 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 4081 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 4141 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 4201 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 4261 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 4321 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 4381 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 4441 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 4501 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 4561 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 4621 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 4681 aggtaactgg cttcagcaga gcgcagatac caaatactat tcttctagtg tagccgtagt 4741 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 4801 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 4861 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 4921 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 4981 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 5041 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 5101 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 5161 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 5221 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 5281 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 5341 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatag 5401 accagccgcg taacctggca aaatcggtta cggttgagta ataaatggat gccctgcgta 5461 agcgggtgtg ggcggacaat aaagtcttaa actgaacaaa atagatctaa actatgacaa 5521 taaagtctta aactagacag aatagttgta aactgaaatc agtccagtta tgctgtgaaa 5581 aagcatactg gacttttgtt atggctaaag caaactcttc attttctgaa gtgcaaattg 5641 cccgtcgtat taaagagggg cgtggccaag ggcatggtaa agactatatt cgcggcgttg 5701 tgacaattta ccgaacaact ccgcggccgg gaagccgatc tcggcttgaa cgaattgtta 5761 ggtggcggta cttgggtcga tatcaaagtg catcacttct tcccgtatgc ccaactttgt 5821 atagagagcc actgcgggat cgtcaccgta atctgcttgc acgtagatca cataagcacc 5881 aagcgcgttg gcctcatgct tgaggagatt gatgagcgcg gtggcaatgc cctgcctccg 5941 gtgctcgccg gagactgcga gatcatagat atagatctca ctacgcggct gctcaaactt 6001 gggcagaacg taagccgcga gagcgccaac aaccgcttct tggtcgaagg cagcaagcgc 6061 gatgaatgtc ttactacgga gcaagttccc gaggtaatcg gagtccggct gatgttggga 6121 gtaggtggct acgtctccga actcacgacc gaaaagatca agagcagccc gcatggattt 6181 gacttggtca gggccgagcc tacatgtgcg aatgatgccc atacttgagc cacctaactt 6241 tgttttaggg cgactgccct gctgcgtaac atcgttgctg ctgcgtaaca tcgttgctgc 6301 tccataacat caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca 6361 tagactgtac aaaaaaacag tcataacaag ccatgaaaac cgccactgcg ccgttaccac 6421 cgctgcgttc ggtcaaggtt ctggaccagt tgcgtgagcg catacgctac ttgcattaca 6481 gtttacgaac cgaacaggct tatgtcaact gggtttgtgc cttcatccgt ttccacggtg 6541 tgcgtcaccc ggcaaccttg ggcagcagcg aagtcgaggc atttctgtcc tggctggcga 6601 acgagcgcaa ggtttcggtc tccacgcatc gtcaggcatt ggcggccttg ctgttcttct 6661 acggcaaggt gctgtgcacg gatctgccct tgcttcagga gatcggtaga cctcggccgt 6721 cgcggcgctt gccggtggtg ctgaccccgg atgaagtggt tcgcatcctc ggttttctgg 6781 aaggcgagca tcgtttgttc gcccaggact ctagctatag ttctagtggt tggctacagc 6841 tttgtttgta ctatcaacag gttgaactgc tgatcaacag atcctctacg cggccgcggt 6901 accataactt cgtatagcat acattatacg aagttatctg gcctaggtta attaaacgct 6961 ctatggtcta aagtttttat cgaggctaac tgaaacacgg aaggagacaa taccggaagg 7021 aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg 7081 ttcataaacg cggggttcgg tcccagggct ggcactctgt caagtgaacg ctctatggtc 7141 taaagattta aatcgaccta ctccggaata ttaatagatc atggagataa ttaaaatgat 7201 aaccatctcg caaataaata agtattttac tgttttcgta acagttttgt aataaaaaaa 7261 cctataaata ttccggatta ttcataccgt cccaccatcg ggcgcggatc cagccaccat 7321 ggctgagaac gtgaccggcc tgttcaagga ctgctccaaa gtgatcaccg gactgcaccc 7381 cactcaggct cctactcacc tgtctgtgga caccaagttc aagaccgagg gcctgtgcgt 7441 ggacatccct ggtatcccta aggacatgac ctaccgtcgt ctgatctcca tgatgggctt 7501 caagatgaac taccaagtga acggctaccc caacatgttc atcacccgtg aagaggctat 7561 ccgtcacgtg cgcgcttgga tcggtttcga cgttgaaggt tgccacgcta ccagggaagc 7621 tgtgggtact aacctgcctc tgcagctggg attctccacc ggtgttaacc tggtggctgt 7681 gcccactggt tacgtggaca cccctaacaa caccgacttc tcccgtgtgt ccgctaagcc 7741 tcctcctggc gaccaattca agcacttgat ccctctgatg tacaagggcc tgccttggaa 7801 cgtcgtgcgt atcaagatcg tgcagatgct gtctgacacc ctgaagaacc tgtccgaccg 7861 tgtggtgttc gtgctgtggg ctcacggttt cgagctgacc tccatgaagt actttgtgaa 7921 gatcggtccc gagaggacct gctgcttgtg cgatagacgt gctacctgct tctctaccgc 7981 ttccgacacc tacgcttgct ggcaccactc catcggcttc gactacgtgt acaacccctt 8041 catgatcgac gtgcagcagt ggggtttcac cggcaacctg cagtctaacc acgacctgta 8101 ctgccaggtg cacggcaacg ctcacgtggc ctcttgtgac gctatcatga cccgttgcct 8161 ggctgtgcac gagtgcttcg tgaagcgtgt ggactggacc atcgagtacc ccatcatcgg 8221 cgacgagctg aagatcaacg ctgcttgcag aaaggtgcag cacatggtgg tcaaggctgc 8281 tctgctggct gacaagttcc ctgtcctgca cgacatcggc aaccccaagg ctattaagtg 8341 cgtgccccag gctgacgtcg agtggaagtt ctacgacgct cagccctgct ccgacaaggc 8401 ttacaagatc gaggaactgt tctactccta cgctacccac tcagacaagt tcaccgacgg 8461 tgtctgcctg ttctggaact gcaatgtgga tcgttacccc gctaactcca tcgtgtgccg 8521 tttcgacacc cgtgtgctgt ccaacctgaa cctgcctggt tgcgacggtg gttccctgta 8581 cgtgaacaag cacgctttcc acactccagc tttcgacaag tccgctttcg tgaacctgaa 8641 gcagctgccc ttcttctact actccgactc tccctgcgag tcccacggaa agcaagtggt 8701 gtccgacatc gattacgtgc ccctgaagtc cgctacctgc atcaccagat gcaacctcgg 8761 tggtgctgtg tgccgtcacc acgctaacga gtaccgtctg tacctggacg cttacaacat 8821 gatgatctcc gctggtttct ccctgtgggt gtacaagcag ttcgacactt acaacctgtg 8881 gaacaccttc accaggctgc agtaaaagct tgtcgagaag tactagagga tcataatcag 8941 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa 9001 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg 9061 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 9121 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatct gatcactgct 9181 tgagcctaga agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca 9241 ccgctgagca ataactatca taaccattta aataaatcta tatctcaatc ggggttacgt 9301 cgatacccca ccgagacccc attgggacca atacgcccgc gtttcttcct tttccccacc 9361 ccaaccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaagccc 9421 tgccatagcc actacgggtt aattaaaaac tatatctcaa tcggggtttt tatcgaggct 9481 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag 9541 acagaataaa acgcacgggt gttgggt //