LOCUS       sequence-246048-        5782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression for affinity purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5782)
  AUTHORS   Murthy AC, Dignon GL, Kan Y, Zerze GH, Parekh SH, Mittal J, Fawzi NL
  TITLE     Molecular interactions underlying liquid-liquid phase separation of 
            the FUS low-complexity domain.
  JOURNAL   Nat Struct Mol Biol. 2019 Jul;26(7):637-648. doi: 
            10.1038/s41594-019-0250-x. Epub 2019 Jul 1.
  PUBMED    31270472
REFERENCE   2  (bases 1 to 5782)
  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/s41594-019-0250-x"; journalName: "Nat Struct Mol Biol"; 
            date: "2019-07"; volume: "26"; issue: "7"; pages: "637-648"
COMMENT     Sequence Label: RP1B FUS 1-163 QQ4xSS #1
FEATURES             Location/Qualifiers
     source          1..5782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     33..52
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     136..155
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        136..154
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    155..179
                     /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             194..216
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             241..258
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             259..279
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             660..671
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             782..799
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(852..870)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      866..913
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      950..1405
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1037..1056)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1247..1268
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1497..2312)
                     /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(1497..2312)
                     /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     2220..2239
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2304..2323
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2434..3022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2923..2942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3176..3193
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3208..3347
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3273..3292)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3433..3455
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3449..3640)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4449..5531)
                     /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     5493..5512
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5532..5609)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
       61 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
      121 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
      181 ctctagaaat aattttgttt aactttaaga aggagatata ccatgggctc tgataaaatt
      241 catcatcatc atcatcacga aaatctttat tttcaaggta tggcctcaaa cgattatacc
      301 caacaagcaa cccaaagcta tggggcctac cccacccagc ccgggcaggg ctattccagc
      361 agcagcagtc agccctacgg acagcagagt tacagtggtt atagccagtc cacggacact
      421 tcaggctatg gccagagcag ctattcttct tatggccaga gccagaacac aggctatgga
      481 actcagtcaa ctccccaggg atatggctcg actggcggct atggcagtag ccagagctcc
      541 caatcgtctt acgggagcag ctcctcctat cctggctatg gccagcagcc agctcccagc
      601 agcacctcgg gaagttacgg tagcagttct cagagcagca gctatgggca gccccagagt
      661 gggagctaca gcagcagccc tagctatggt ggacagcagc aaagctatgg acagcagcaa
      721 agctataatc cccctcaggg ctatggaagc agcaaccagt acaacagctg agatcctcga
      781 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
      841 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
      901 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
      961 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     1021 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1081 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     1141 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     1201 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     1261 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     1321 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     1381 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1441 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
     1501 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     1561 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     1621 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     1681 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     1741 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     1801 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     1861 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     1921 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     1981 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     2041 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     2101 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     2161 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     2221 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     2281 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     2341 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2401 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2461 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2521 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2581 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2641 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2701 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2761 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2821 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2881 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2941 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3001 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3061 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3121 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3181 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     3241 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3301 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     3361 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3421 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3481 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     3541 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     3601 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     3661 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     3721 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     3781 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     3841 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     3901 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     3961 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     4021 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     4081 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     4141 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     4201 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     4261 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     4321 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4381 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4441 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4501 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4561 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4621 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     4681 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     4741 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     4801 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     4861 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     4921 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     4981 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5041 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5101 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5161 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5221 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5281 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5341 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5401 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5461 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5521 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5581 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     5641 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     5701 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     5761 cacgccgaaa caagcgctca tg
//
