LOCUS       sequence-189255-        7284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MBP-tagged phosphomimetic FUS AA 1-163.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7284)
  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 7284)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "EMBO J. 
            2017 Aug 8. pii: e201696394. doi: 10.15252/embj.201696394."
COMMENT     Sequence Label: MBP-FUS_LC_12E
FEATURES             Location/Qualifiers
     source          1..7284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     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             2149..2166
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2219..2237)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2233..2280
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2317..2772
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2404..2423)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2614..2635
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2864..3679)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2864..3679)
                     /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     3587..3606
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3671..3690
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3801..4389
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4290..4309
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4543..4560
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4575..4714
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4640..4659)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4800..4822
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4816..5007)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5816..6898)
                     /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     6860..6879
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6899..6976)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7182..7201
                     /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 agggtatggc ctcaaacgat
     1321 tatgagcaac aagcagagca aagctatggg gcctaccccg agcagcccgg gcagggctat
     1381 gagcagcaga gcgagcagcc ctacggacag cagagttaca gtggttatga gcagtccacg
     1441 gacacttcag gctatggcca gagcagctat tcttcttatg gccaggagca gaacacaggc
     1501 tatggagagc agtcaactcc ccagggatat ggctcgactg gcggctatgg cagtgagcag
     1561 agcgagcaat cgtcttacgg gcagcagtcc tcctatcctg gctatggcca gcagccagct
     1621 cccagcagca cctcgggaag ttacggtagc agtgagcaga gcagcagcta tgggcagccc
     1681 cagagtggga gctacgagca gcagcctagc tatggtggac agcagcaaag ctatggacag
     1741 cagcaaagct ataatccccc tcagggctat ggacagcaga accagtacaa cagctagagt
     1801 ggtggtggag gtggaggaca gcagcgagct ggtgactgga agtgtcctaa tcccacctgt
     1861 gagaatatga acttctcttg gaggaatgaa tgcaaccagt gtaaggcccc taaaccagat
     1921 ggcccaggag ggggaccagg tggctctcac atggggggta actacgggga tgatcgtcgt
     1981 ggtggcagag gaggctatga tcgaggcggc taccggggcc gcggcgggga ccgtggaggc
     2041 ttccgagggg gccggggtgg tggggacaga ggtggctttg gccctggcaa gatggattcc
     2101 aggggtgagc acagacagga tcgcagggag aggccgtatt gactcgagca ccaccaccac
     2161 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     2221 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2281 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     2341 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2401 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2461 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2521 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2581 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2641 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2701 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2761 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2821 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     2881 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     2941 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     3001 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     3061 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     3121 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     3181 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     3241 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     3301 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     3361 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     3421 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     3481 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     3541 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     3601 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     3661 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     3721 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3781 gaaaagatca aaggatcttc ttgatatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3841 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3901 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3961 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4021 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4081 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4141 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4201 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4261 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4321 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4381 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4441 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4501 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4561 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4621 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4681 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4741 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4801 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4861 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4921 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4981 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5041 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5101 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5161 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5221 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5281 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5341 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5401 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5461 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     5521 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     5581 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     5641 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     5701 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5761 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5821 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5881 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     5941 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     6001 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     6061 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     6121 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     6181 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     6241 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     6301 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     6361 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     6421 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     6481 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     6541 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     6601 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     6661 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     6721 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     6781 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6841 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     6901 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     6961 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     7021 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     7081 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     7141 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     7201 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     7261 atcgagatct cgatcccgcg aaat
//
