LOCUS       sequence-450205-        5778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4 serines to alanine at residue positions 70, 84, 89, 95.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5778)
  AUTHORS   Wake N, Weng SL, Zheng T, Wang SH, Kirilenko V, Mittal J, Fawzi NL
  TITLE     Expanding the molecular grammar of polar residues and arginine in 
            FUS phase separation.
  JOURNAL   Nat Chem Biol. 2025 Jul;21(7):1076-1088. doi: 
            10.1038/s41589-024-01828-6. Epub 2025 Feb 7.
  PUBMED    39920231
REFERENCE   2  (bases 1 to 5778)
  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"; doi: 
            "10.1038/s41589-024-01828-6"; journalName: "Nat Chem Biol"; date: 
            "2025-07"; volume: "21"; issue: "7"; pages: "1076-1088"
COMMENT     Sequence Label: FUS LC 4S->A#2
FEATURES             Location/Qualifiers
     source          1..5778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..106
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(335..1150)
                     /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(335..1150)
                     /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     1058..1077
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1142..1161
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1272..1860
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1761..1780
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2014..2031
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2046..2185
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2111..2130)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2271..2293
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2287..2478)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3287..4369)
                     /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     4331..4350
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4370..4447)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4653..4672
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4756..4775
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4756..4774
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4775..4799
                     /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             4814..4836
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4861..4878
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4879..4899
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5398..5415
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5468..5486)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5482..5529
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5566..5778,1..243)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5653..5672)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
       61 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
      121 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
      181 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
      241 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
      301 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
      361 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
      421 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
      481 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
      541 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
      601 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
      661 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
      721 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
      781 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
      841 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
      901 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
      961 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     1021 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     1081 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     1141 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     1201 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1261 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1321 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1381 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     1441 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1501 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1561 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1621 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1681 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1741 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1801 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1861 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     1921 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     1981 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     2041 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     2101 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     2161 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     2221 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     2281 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     2341 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     2401 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     2461 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     2521 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     2581 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     2641 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     2701 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     2761 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     2821 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     2881 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     2941 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     3001 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     3061 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     3121 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     3181 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     3241 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     3301 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3361 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     3421 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     3481 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     3541 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     3601 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     3661 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     3721 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     3781 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     3841 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     3901 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     3961 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     4021 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     4081 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     4141 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     4201 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     4261 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     4321 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     4381 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     4441 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     4501 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     4561 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     4621 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     4681 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     4741 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     4801 ctctagaaat aattttgttt aactttaaga aggagatata ccatgggctc tgataaaatt
     4861 catcatcatc atcatcacga aaatctttat tttcaaggta tggcgagcaa cgactacacc
     4921 cagcaagcga cccagagcta cggtgcgtat ccgacccagc cgggtcaagg ctacagccag
     4981 caaagcagcc agccgtatgg tcagcaaagc tacagcggct atagccagag caccgatacc
     5041 agcggttacg gccagagcag ctacagcagc tatggccaga gccaaaacac cggttatggt
     5101 acccaagcga ccccgcaagg ttatggtagc accggtggct atggtagcgc gcagagcagc
     5161 caagcgagct acggccagca agcgagctat ccgggttatg gtcaacagcc ggcgccgagc
     5221 agcaccagcg gtagctacgg tagcagcagc cagagcagca gctacggtca gccgcaaagc
     5281 ggcagctaca gccagcaacc gagctatggt ggccagcaac agagctacgg tcaacagcaa
     5341 agctataacc cgccgcaggg ttacggccag caaaaccaat ataacagctg actcgagcac
     5401 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     5461 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     5521 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     5581 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     5641 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     5701 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     5761 tcgaccccaa aaaacttg
//
