LOCUS Exported 7491 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION express MBP tagged SARS-CoV-2 Nucleocapsid protein without the linker. ACCESSION . VERSION . KEYWORDS SARS-CoV-2_N_deltalinker SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7491) AUTHORS Perdikari TM, Murthy AC, Ryan VH, Watters S, Naik MT, Fawzi NL TITLE SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs. JOURNAL EMBO J. 2020 Nov 17:e106478. doi: 10.15252/embj.2020106478. PUBMED 33200826 REFERENCE 2 (bases 1 to 7491) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7491 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /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 59..81 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 106..123 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 136..1233 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQT" regulatory 1090..1099 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 1207..1230 /label=MBP-F /note="Maltose binding protein, forward primer" protein_bind 1252..1276 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 1285..1305 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2356..2373 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2426..2444) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2440..2487 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2524..2979 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2611..2630) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2821..2842 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3071..3886) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 3794..3813 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 3878..3897 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4008..4596 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4497..4516 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4750..4767 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4782..4921 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4847..4866) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5007..5029 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5023..5214) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(6023..7105) /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 7067..7086 /label=LacI-R /note="LacI, reverse primer" promoter complement(7106..7183) /gene="lacI" /label=lacI promoter primer_bind 7389..7408 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atataccatg ggatctgata aaattcatca tcatcatcat 121 cacagcagcg gtaccaaaat cgaagaaggt aaactggtaa tctggattaa cggcgataaa 181 ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc 241 accgttgagc atccggataa actggaagag aaattcccac aggttgcggc aactggcgat 301 ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg 361 ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat 421 gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg 481 atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg 541 gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac 601 ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag 661 tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg 721 gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct 781 gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc 841 gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc 901 aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg 961 gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa 1021 gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggc gaaagatcca 1081 cgtattgccg ccaccatgga aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag 1141 atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag 1201 actgtcgatg aagccctgaa agacgcgcag actaattcgg gatctgatat cacaagtttg 1261 tacaaaaaag ctgaaggcgg taccgagaac ctgtacttcc agggtcatat gagcgacaat 1321 ggtccgcaga atcaacgtaa tgcgccgcgt atcacctttg gtggtccgag cgacagcacc 1381 ggcagcaatc agaatggcga acgtagcggt gcgcgtagca aacagcgtcg tccgcagggt 1441 ctgccgaaca acaccgcgag ctggtttacc gcgctgaccc aacacggcaa agaagacctg 1501 aaattcccgc gtggtcaagg cgttccgatc aacaccaaca gcagcccgga tgaccagatt 1561 ggctattacc gtcgtgcgac ccgtcgtatt cgtggcggtg atggcaagat gaaagacctg 1621 agcccgcgtt ggtatttcta ctatctgggt accggcccgg aggcgggtct gccgtatggt 1681 gcgaacaagg acggtatcat ttgggtggcg accgaaggcg cgctgaacac cccgaaggac 1741 cacatcggta cccgtaaccc ggcgaacaac gcggcgatcg ttctgcagct gccgcaaggt 1801 accaccctgc cgaaaggttt ctatgcggag aagaaaagcg cggcggaggc gagcaaaaag 1861 ccgcgtcaga aacgtaccgc gaccaaagcg tacaacgtga cccaagcgtt tggtcgtcgt 1921 ggtccggaac aaacccaggg taacttcggt gatcaggaac tgattcgtca aggtaccgac 1981 tacaaacact ggccgcaaat cgcgcagttc gcgccgagcg cgagcgcgtt ctttggtatg 2041 agccgtatcg gtatggaagt taccccgagc ggtacctggc tgacctatac cggtgcgatc 2101 aagctggacg ataaggatcc gaacttcaag gaccaggtta tcctgctgaa caagcacatc 2161 gacgcgtaca aaaccttccc gccgaccgag ccgaagaaag acaagaagaa gaaagcggac 2221 gagacccaag cgctgccgca acgtcagaaa aagcaacaga ccgttaccct gctgccggcg 2281 gcggatctgg acgactttag caaacaactg caacagagca tgagcagcgc ggatagcacc 2341 caggcgtaac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga 2401 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc 2461 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat 2521 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 2581 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 2641 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 2701 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 2761 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 2821 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 2881 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 2941 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa 3001 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 3061 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag 3121 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga 3181 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat 3241 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat 3301 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt 3361 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca 3421 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa 3481 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg 3541 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta 3601 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg 3661 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat 3721 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg 3781 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat 3841 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat 3901 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt 3961 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg atatcctttt 4021 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 4081 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 4141 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 4201 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 4261 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 4321 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 4381 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 4441 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 4501 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 4561 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 4621 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 4681 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 4741 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc 4801 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg 4861 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc 4921 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 4981 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 5041 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc 5101 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt 5161 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc 5221 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat 5281 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa 5341 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg 5401 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat 5461 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa 5521 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca 5581 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag 5641 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat 5701 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag 5761 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa 5821 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat 5881 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct 5941 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta 6001 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 6061 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg 6121 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct 6181 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga 6241 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg 6301 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca 6361 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg 6421 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt 6481 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta 6541 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca 6601 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat 6661 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat 6721 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg 6781 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt 6841 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg 6901 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg 6961 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt 7021 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga 7081 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct 7141 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct 7201 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc 7261 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg 7321 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct 7381 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg 7441 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t //