LOCUS Exported 5936 bp ds-DNA circular SYN 28-JUL-2021 DEFINITION Expression of 6xHis tagged protein for crystallization, includes TEV protease site for tag removal. ACCESSION . VERSION . KEYWORDS Nsp3 Mac1 - P43 (Oxford) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5936) AUTHORS Schuller M, Correy GJ, Gahbauer S, Fearon D, Wu T, Diaz RE, Young ID, Carvalho Martins L, Smith DH, Schulze-Gahmen U, Owens TW, Deshpande I, Merz GE, Thwin AC, Biel JT, Peters JK, Moritz M, Herrera N, Kratochvil HT, Aimon A, Bennett JM, Brandao Neto J, Cohen AE, Dias A, Douangamath A, Dunnett L, Fedorov O, Ferla MP, Fuchs MR, Gorrie-Stone TJ, Holton JM, Johnson MG, Krojer T, Meigs G, Powell AJ, Rack JGM, Rangel VL, Russi S, Skyner RE, Smith CA, Soares AS, Wierman JL, Zhu K, O'Brien P, Jura N, Ashworth A, Irwin JJ, Thompson MC, Gestwicki JE, von Delft F, Shoichet BK, Fraser JS, Ahel I TITLE Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking. JOURNAL Sci Adv. 2021 Apr 14;7(16). pii: 7/16/eabf8711. doi: 10.1126/sciadv.abf8711. Print 2021 Apr. PUBMED 33853786 REFERENCE 2 (bases 1 to 5936) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 28, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5936 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 7..24 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 49..69 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 583..600 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(653..671) /label=T7 Term /note="T7 terminator, reverse primer" terminator 667..714 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 751..1206 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(838..857) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1048..1069 /label=F1ori-F /note="F1 origin, forward primer" promoter 1232..1336 /gene="bla" /label=AmpR promoter CDS 1337..2197 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1555..1574) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2368..2956 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2857..2876 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3110..3127 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3142..3281 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3207..3226) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3367..3389 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3383..3574) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4383..5465) /codon_start=1 /gene="lacI" /product="lac repressor" /label=lacI /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 5427..5446 /label=LacI-R /note="LacI, reverse primer" promoter complement(5466..5543) /gene="lacI" /label=lacI promoter primer_bind 5749..5768 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 5852..5871 /label=T7 /note="T7 promoter, forward primer" promoter 5852..5870 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5871..5895 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." RBS 5910..5932 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" ORIGIN 1 catatgcacc atcaccatca ccattcttct ggtgtagatc tgggtaccga aaacctgtat 61 tttcagtcga tggtaaattc tttcagtggt tatctgaagt tgacggataa cgtctatatt 121 aaaaacgctg acattgtgga agaggcgaaa aaggtaaagc ctacagttgt ggttaatgcg 181 gcaaacgtct atttaaaaca tggtggaggt gtggcggggg ctctcaacaa agcgactaac 241 aatgcaatgc aggtggaaag cgatgattac attgcgacca acggcccact gaaagttggc 301 ggttcgtgcg tgctctcagg tcacaatctg gctaaacatt gtctgcacgt cgtggggccc 361 aacgttaata aaggcgagga tatccaacta ttaaaaagcg catacgaaaa ctttaaccag 421 catgaagttc tgctggcgcc gttgcttagc gccggaatat tcggcgccga cccgatccac 481 tccctgcgcg tctgcgttga tacggtacgt accaatgtgt atctggccgt ctttgacaaa 541 aatctttacg ataaactggt gagttccttt ctctgactcg agcaccacca ccaccaccac 601 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag 661 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 721 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg 781 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 841 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 901 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 961 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 1021 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 1081 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 1141 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta 1201 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 1261 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1321 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1381 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1441 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1501 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1561 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1621 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1681 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1741 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1801 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1861 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga 1921 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 1981 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 2041 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 2101 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 2161 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 2221 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 2281 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2341 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2401 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2461 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 2521 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2581 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2641 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2701 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 2761 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2821 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2881 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 2941 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 3001 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 3061 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 3121 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 3181 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag 3241 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac 3301 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 3361 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc 3421 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat 3481 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg 3541 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc 3601 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg 3661 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc 3721 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag 3781 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg 3841 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg 3901 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg 3961 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga 4021 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga 4081 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata 4141 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga 4201 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg 4261 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca 4321 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc 4381 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4441 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt 4501 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg 4561 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata 4621 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc 4681 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc 4741 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg 4801 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta 4861 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg 4921 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg 4981 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca 5041 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc 5101 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg 5161 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc 5221 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc 5281 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc 5341 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg 5401 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt 5461 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag 5521 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat 5581 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc 5641 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc 5701 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc 5761 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc 5821 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga 5881 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatata //