LOCUS Exported 5157 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression of Nsp1 residues 13-127. ACCESSION . VERSION . KEYWORDS pMCSG53_Nsp1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5157) AUTHORS Semper C, Watanabe N, Savchenko A TITLE Structural characterization of nonstructural protein 1 from SARS-CoV-2. JOURNAL iScience. 2021 Jan 22;24(1):101903. doi: 10.1016/j.isci.2020.101903. Epub 2020 Dec 7. PUBMED 33319167 REFERENCE 2 (bases 1 to 5157) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5157 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 970..989 /label=LacI-R /note="LacI, reverse primer" promoter complement(1009..1086) /gene="lacI" /label=lacI promoter tRNA 1226..1301 /gene="ileX" /product="E. coli tRNA-Ile recognizing AUA" /label=ileX primer_bind 1450..1469 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" tRNA 1571..1647 /gene="argU" /product="E. coli tRNA-Arg recognizing AGA and AGG" /label=argU primer_bind 1922..1941 /label=T7 /note="T7 promoter, forward primer" promoter 1922..1940 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1941..1965 /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 1980..2002 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 2013..2030 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 2055..2075 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 2493..2510 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2563..2581) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2577..2624 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind complement(2748..2767) /label=F1ori-R /note="F1 origin, reverse primer" promoter 2934..3037 /gene="bla" /label=AmpR promoter CDS 3038..3898 /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(3256..3275) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4069..4657 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4558..4577 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4811..4828 /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(4908..4927) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" CDS complement(5083..1008) /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" ORIGIN 1 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg 61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc 121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc 181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat 241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat 301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc 361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg 421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa 481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt 541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc 601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg 661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga 721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga 781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc 841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt 901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata 961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct 1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat 1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta 1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcgga ttgcgacacg 1201 gagttacttt ataatccgct accatggccc cttagctcag tggttagagc aggcgactca 1261 taatcgcttg gtcgctggtt caagtccagc aggggccacc agatatagca aaggctgacg 1321 agaaatcgtc agcctttttg catgcaagga gatggcgccc aacagtcccc cggccacggg 1381 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc 1441 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggcgag 1501 atcaaaagcc attgactcag caagggttga ccgtataatt cacgcgatta caccgcattg 1561 cggtatcaac gcgcccttag ctcagttgga tagagcaacg accttctaag tcgtgggccg 1621 caggttcgaa tcctgcaggg cgcgccatta caattcaatc agttacgcct tctttatatc 1681 ctccataatt ccagagtggg acatatttgg gacattatca ccaaaaatgt cgtctatttt 1741 cctcgcatgc tctgtcaaat gattaggcgc aaggtgagca tacctacgaa ccatttctat 1801 ggactcccat ccgcccattt cctgaagcac tgataatggg acgcctgact gaatccaccg 1861 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa 1921 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt 1981 ttgtttaact ttaagaagga gatatacata tgcaccatca tcatcatcat tcttctggtg 2041 tagatctggg taccgagaac ctgtacttcc aatccaatgc ccatgttcag ctgagtttac 2101 cggttctgca agttcgtgat gttctggttc gcggttttgg tgatagcgtt gaagaagttc 2161 tgagtgaagc acgtcaacac ctgaaagatg gtacatgcgg cttagtggaa gtggaaaaag 2221 gtgttttacc tcagctggaa cagccgtacg tgttcattaa acgcagcgat gcaagaacag 2281 cacctcatgg tcatgttatg gttgaactgg tggcagaact ggaaggtatc cagtatggta 2341 gatctggtga aacactgggt gttttagttc cgcatgtggg cgaaattcct gtggcatacc 2401 gtaaagtgct gctgcgtaaa aatggctaac attggaagtg gataacggat ccgaattcga 2461 gctccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg 2521 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag 2581 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta 2641 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 2701 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 2761 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 2821 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 2881 tgatggttca cattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa 2941 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 3001 cctgataaat gcttcaataa tttgaaaaag gaagagtatg agtattcaac atttccgtgt 3061 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 3121 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 3181 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 3241 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 3301 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 3361 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 3421 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 3481 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 3541 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt 3601 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 3661 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 3721 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg 3781 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 3841 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 3901 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 3961 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 4021 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 4081 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 4141 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 4201 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt 4261 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 4321 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 4381 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 4441 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 4501 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 4561 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 4621 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 4681 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 4741 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac 4801 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct 4861 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct 4921 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgt agtcatgccc 4981 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc 5041 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag 5101 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggc //