LOCUS       sequence-450267-        5778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7S / Q160S double mutation .
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 noYQ_noYT
FEATURES             Location/Qualifiers
     source          1..5778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     84..105
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(334..1149)
                     /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(334..1149)
                     /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     1057..1076
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1141..1160
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1271..1859
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1760..1779
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2013..2030
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2045..2184
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2110..2129)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2270..2292
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2286..2477)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3286..4368)
                     /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     4330..4349
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4369..4446)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4652..4671
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4755..4774
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4755..4773
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4774..4798
                     /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             4813..4835
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4860..4877
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4878..4898
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5397..5414
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5467..5485)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5481..5528
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5565..5778,1..242)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5652..5671)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
       61 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
      121 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
      181 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
      241 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
      301 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
      361 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
      421 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
      481 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
      541 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
      601 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
      661 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
      721 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
      781 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
      841 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
      901 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
      961 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     1021 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     1081 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     1141 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     1201 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     1261 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     1321 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1381 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     1441 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1501 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     1561 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     1621 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     1681 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     1741 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     1801 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     1861 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     1921 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     1981 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2041 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     2101 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     2161 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     2221 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     2281 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     2341 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     2401 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     2461 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     2521 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     2581 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     2641 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     2701 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     2761 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     2821 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     2881 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     2941 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     3001 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     3061 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     3121 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     3181 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     3241 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     3301 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     3361 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     3421 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     3481 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     3541 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     3601 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     3661 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     3721 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     3781 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     3841 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     3901 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     3961 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     4021 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     4081 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     4141 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     4201 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     4261 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     4321 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     4381 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     4441 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     4501 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     4561 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     4621 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     4681 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     4741 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     4801 tctagaaata attttgttta actttaagaa ggagatatac catgggctct gataaaattc
     4861 atcaccatca ccatcacgaa aatctttatt ttcaaggtat ggcgagcaac gactacagcc
     4921 agcaagcgac ccagagctac ggtgcgtatc cgacccagcc gggtcaaggc tacagccagc
     4981 aaagcagcca gccgtatggt cagcaaagct acagcggcta tagccagagc accgatacca
     5041 gcggttacgg ccaaagcagc tacagcagct atggccagag ccaaaacacc ggttatggca
     5101 cccagagcac cccgcaaggt tacggcagca ccggtggcta tggtagcagc cagagcagcc
     5161 aaagcagcta cggccagcaa agcagctatc cgggttatgg tcaacagccg gcgccgagca
     5221 gcaccagcgg tagctacggt agcagcagcc agagcagcag ctacggtcag ccgcaaagcg
     5281 gcagctacag ccagcaaccg agctatggtg gccagcaaca gagctacggt caacagcaaa
     5341 gctataaccc gccgcagggt tacggccagc aaaacagcta taacagctaa ctcgagcacc
     5401 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     5461 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     5521 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     5581 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5641 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5701 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5761 cgaccccaaa aaacttga
//
