LOCUS       sequence-488769-        6678 bp    DNA     circular SYN 08-OCT-2025
DEFINITION  Expresses MBP-tagged FUS RGG3 domain in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6678)
  AUTHORS   Murthy AC, Tang WS, Jovic N, Janke AM, Seo DH, Perdikari TM, Mittal 
            J, Fawzi NL
  TITLE     Molecular interactions contributing to FUS SYGQ LC-RGG phase 
            separation and co-partitioning with RNA polymerase II heptads.
  JOURNAL   Nat Struct Mol Biol. 2021 Nov;28(11):923-935. doi: 
            10.1038/s41594-021-00677-4. Epub 2021 Nov 10.
  PUBMED    34759379
REFERENCE   2  (bases 1 to 6678)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41594-021-00677-4"; journalName: "Nat Struct Mol Biol"; 
            date: "2021-11"; volume: "28"; issue: "11"; pages: "923-935"
COMMENT     Sequence Label: MBP-FUS-RGG3
FEATURES             Location/Qualifiers
     source          1..6678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(170..190)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(199..223)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(242..1339)
                     /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"
     primer_bind     complement(245..268)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      376..385
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1352..1369)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1394..1416
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1431..1455
                     /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)."
     primer_bind     complement(1455..1474)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1456..1474)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1558..1577)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1783..1860
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1861..2943
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLASWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1880..1899)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3752..3943
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3937..3959)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4045..4184
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4100..4119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4199..4216)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4370..4958)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4450..4469)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5069..5088)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5080..5895
                     /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             5080..5895
                     /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     complement(5153..5172)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5987..6442)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6124..6145)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6336..6355
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6479..6526)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6522..6540
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6593..6610)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 caaagccacc tctgtcccca ccaccccggc cccctcggaa gcctccacgg tccccgccgc
       61 ggccccggta gccgcctcga tcatagcctc ctctgccacc acgacgatca tccccgtagt
      121 taccccccat gtgagagcca cctggtcccc ctcctgggcc atccatatga ccctggaagt
      181 acaggttctc ggtaccgcct tcagcttttt tgtacaaact tgtgatatca gatcccgaat
      241 tagtctgcgc gtctttcagg gcttcatcga cagtctgacg accgctggcg gcgttgatca
      301 ccgcagtacg cacggcatac cagaaagcgg acatctgcgg gatgttcggc atgatttcac
      361 ctttctgggc gttttccatg gtggcggcaa tacgtggatc tttcgccaac tcttcctcgt
      421 aagacttcag cgctacggca cccagcggtt tgtctttatt aaccgcttcc agaccttcat
      481 cagtcagcag atagttttcg aggaactctt ttgccagctc tttgttcgga ctggcggcgt
      541 taatacctgc gctcagcacg ccaacgaacg gtttggatgg ttgacccttg aaggtcggca
      601 gtaccgttac accataattc actttgctgg tgtcgatgtt ggaccatgcc cacgggccgt
      661 tgatggtcat cgctgtttcg cctttattaa aggcagcttc tgcgatggag taatcggtgt
      721 ctgcattcat gtgtttgttt ttaatcaggt caaccaggaa ggtcagaccc gctttcgcgc
      781 cagcgttatc cacgcccacg tctttaatgt cgtacttgcc gttttcatac ttgaacgcat
      841 aacccccgtc agcagcaatc agcggccagg tgaagtacgg ttcttgcagg ttgaacatca
      901 gcgcgctctt acctttcgct ttcagttctt tatccagcgc cgggatctct tcccaggttt
      961 ttggcgggtt cggcagcaga tctttgttat aaatcagcga taacgcttca acagcgatcg
     1021 ggtaagcaat cagcttgccg ttgtaacgta cggcatccca ggtaaacgga tacagcttgt
     1081 cctggaacgc tttgtccggg gtgatttcag ccaacaggcc agattgagcg tagccaccaa
     1141 agcggtcgtg tgcccagaag ataatgtcag ggccatcgcc agttgccgca acctgtggga
     1201 atttctcttc cagtttatcc ggatgctcaa cggtgacttt aattccggta tctttctcga
     1261 atttcttacc gacttcagcg agaccgttat agcctttatc gccgttaatc cagattacca
     1321 gtttaccttc ttcgattttg gtaccgctgc tgtgatgatg atgatgatga attttatcag
     1381 atcccatggt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
     1441 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
     1501 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
     1561 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
     1621 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
     1681 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
     1741 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
     1801 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     1861 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     1921 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     1981 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     2041 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     2101 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     2161 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     2221 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     2281 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     2341 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     2401 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctagc
     2461 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     2521 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     2581 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     2641 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     2701 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     2761 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     2821 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     2881 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2941 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat
     3001 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     3061 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     3121 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     3181 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     3241 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat
     3301 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     3361 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     3421 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     3481 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     3541 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     3601 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     3661 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     3721 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     3781 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     3841 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     3901 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3961 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     4021 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     4081 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt
     4141 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     4201 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4261 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4321 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4381 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4441 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4501 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4561 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4621 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4681 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4741 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4801 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4861 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4921 gcaagcagca gattacgcgc agaaaaaaag gatatcaaga agatcctttg atcttttcta
     4981 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata
     5041 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     5101 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     5161 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     5221 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     5281 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     5341 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     5401 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     5461 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     5521 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     5581 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     5641 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     5701 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     5761 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     5821 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     5881 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     5941 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     6001 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     6061 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     6121 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     6181 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     6241 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6301 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     6361 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     6421 aatgcgccgc tacagggcgc gtcccattcg ccaatccgga tatagttcct cctttcagca
     6481 aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat tgctcagcgg
     6541 tggcagcagc caactcagct tcctttcggg ctttgttagc agccggatct cagtggtggt
     6601 ggtggtggtg ctcctaatac ggcctctccc tgcgatcctg tctgtgctca cccctggaat
     6661 ccatcttgcc aggttagc
//
