LOCUS Exported 8031 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses MBP-tagged full length phosphomimetic FUS. ACCESSION . VERSION . KEYWORDS MBP-FUS_FL_12E SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8031) AUTHORS Monahan Z, Ryan VH, Janke AM, Burke KA, Rhoads SN, Zerze GH, O'Meally R, Dignon GL, Conicella AE, Zheng W, Best RB, Cole RN, Mittal J, Shewmaker F, Fawzi NL TITLE Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity. JOURNAL EMBO J. 2017 Aug 8. pii: e201696394. doi: 10.15252/embj.201696394. PUBMED 28790177 REFERENCE 2 (bases 1 to 8031) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8031 /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 2896..2913 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2966..2984) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2980..3027 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3064..3519 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3151..3170) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3361..3382 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3611..4426) /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 4334..4353 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4418..4437 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4548..5136 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5037..5056 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5290..5307 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5322..5461 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5387..5406) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5547..5569 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5563..5754) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(6563..7645) /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 7607..7626 /label=LacI-R /note="LacI, reverse primer" promoter complement(7646..7723) /gene="lacI" /label=lacI promoter primer_bind 7929..7948 /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 ggcctcaaac 1321 gattatgagc aacaagcaga gcaaagctat ggggcctacc ccgagcagcc cgggcagggc 1381 tatgagcagc agagcgagca gccctacgga cagcagagtt acagtggtta tgagcagtcc 1441 acggacactt caggctatgg ccagagcagc tattcttctt atggccagga gcagaacaca 1501 ggctatggag agcagtcaac tccccaggga tatggctcga ctggcggcta tggcagtgag 1561 cagagcgagc aatcgtctta cgggcagcag tcctcctatc ctggctatgg ccagcagcca 1621 gctcccagca gcacctcggg aagttacggt agcagtgagc agagcagcag ctatgggcag 1681 ccccagagtg ggagctacga gcagcagcct agctatggtg gacagcagca aagctatgga 1741 cagcagcaaa gctataatcc ccctcagggc tatggacagc agaaccagta caacagcagc 1801 agtggtggtg gaggtggagg tggaggtgga ggtaactatg gccaagatca atcctccatg 1861 agtagtggtg gtggcagtgg tggcggttat ggcaatcaag accagagtgg tggaggtggc 1921 agcggtggct atggacagca ggaccgtgga ggccgcggca ggggtggcag tggtggcggc 1981 ggcggcggcg gcggtggtgg ttacaaccgc agcagtggtg gctatgaacc cagaggtcgt 2041 ggaggtggcc gtggaggcag aggtggcatg ggcggaagtg accgtggtgg cttcaataaa 2101 tttggtggcc ctcgggacca aggatcacgt catgactccg aacaggataa ttcagacaac 2161 aacaccatct ttgtgcaagg cctgggtgag aatgttacaa ttgagtctgt ggctgattac 2221 ttcaagcaga ttggtattat taagacaaac aagaaaacgg gacagcccat gattaatttg 2281 tacacagaca gggaaactgg caagctgaag ggagaggcaa cggtctcttt tgatgaccca 2341 ccttcagcta aagcagctat tgactggttt gatggtaaag aattctccgg aaatcctatc 2401 aaggtctcat ttgctactcg ccgggcagac tttaatcggg gtggtggcaa tggtcgtgga 2461 ggccgagggc gaggaggacc catgggccgt ggaggctatg gaggtggtgg cagtggtggt 2521 ggtggccgag gaggatttcc cagtggaggt ggtggcggtg gaggacagca gcgagctggt 2581 gactggaagt gtcctaatcc cacctgtgag aatatgaact tctcttggag gaatgaatgc 2641 aaccagtgta aggcccctaa accagatggc ccaggagggg gaccaggtgg ctctcacatg 2701 gggggtaact acggggatga tcgtcgtggt ggcagaggag gctatgatcg aggcggctac 2761 cggggccgcg gcggggaccg tggaggcttc cgagggggcc ggggtggtgg ggacagaggt 2821 ggctttggcc ctggcaagat ggattccagg ggtgagcaca gacaggatcg cagggagagg 2881 ccgtattgac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga 2941 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc 3001 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat 3061 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 3121 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 3181 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 3241 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 3301 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 3361 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 3421 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 3481 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa 3541 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 3601 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag 3661 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga 3721 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat 3781 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat 3841 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt 3901 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca 3961 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa 4021 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg 4081 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta 4141 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg 4201 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat 4261 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg 4321 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat 4381 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat 4441 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt 4501 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg atatcctttt 4561 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 4621 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 4681 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 4741 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 4801 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 4861 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 4921 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 4981 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 5041 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 5101 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 5161 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 5221 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 5281 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc 5341 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg 5401 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc 5461 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 5521 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 5581 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc 5641 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt 5701 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc 5761 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat 5821 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa 5881 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg 5941 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat 6001 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa 6061 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca 6121 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag 6181 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat 6241 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag 6301 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa 6361 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat 6421 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct 6481 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta 6541 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 6601 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg 6661 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct 6721 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga 6781 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg 6841 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca 6901 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg 6961 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt 7021 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta 7081 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca 7141 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat 7201 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat 7261 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg 7321 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt 7381 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg 7441 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg 7501 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt 7561 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga 7621 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct 7681 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct 7741 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc 7801 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg 7861 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct 7921 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg 7981 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t //