LOCUS       sequence-246052-        5778 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 5778)
  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 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/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 #2
FEATURES             Location/Qualifiers
     source          1..5778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..61
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     145..164
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        145..163
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    164..188
                     /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             203..225
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             250..267
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             268..288
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             669..680
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             787..804
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(857..875)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      871..918
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      955..1410
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1042..1061)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1252..1273
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1502..2317)
                     /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(1502..2317)
                     /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     2225..2244
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2309..2328
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2439..3027
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2928..2947
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3181..3198
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3213..3352
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3278..3297)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3438..3460
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3454..3645)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4454..5536)
                     /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     5498..5517
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5537..5614)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
       61 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
      121 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
      181 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac catgggctct
      241 gataaaattc atcaccatca ccatcacgaa aatctttatt ttcaaggtat ggcctcaaac
      301 gattatacca gcagcgcaac ccaaagctat ggggcctacc ccacccagcc cgggcagggc
      361 tattcccagc agagcagtca gccctacgga cagcagagtt acagtggtta tagccagtcc
      421 acggacactt caggctatgg ccagagcagc tattcttctt atggcagcag cagcaacaca
      481 ggctatggaa ctcagtcaac tccccaggga tatggctcga ctggcggcta tggcagtagc
      541 cagagctccc aatcgtctta cgggcagcag tcctcctatc ctggctatgg cagcagccca
      601 gctcccagca gcacctcggg aagttacggt agcagttctc agagcagcag ctatgggcag
      661 ccccagagtg ggagctacag ccagcagcct agctatggtg gaagcagcca aagctatgga
      721 cagcagcaaa gctataatcc ccctcagggc tatggacagc agaaccagta caacagctga
      781 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
      841 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      901 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
      961 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1021 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1081 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1141 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1201 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1261 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1321 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1381 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     1441 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     1501 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     1561 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     1621 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     1681 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     1741 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     1801 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     1861 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     1921 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     1981 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     2041 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     2101 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     2161 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     2221 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     2281 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     2341 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2401 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2461 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2521 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2581 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2641 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2701 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2761 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2821 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2881 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2941 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3001 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3061 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3121 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3181 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     3241 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     3301 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     3361 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3421 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3481 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     3541 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     3601 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     3661 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     3721 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     3781 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     3841 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     3901 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     3961 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     4021 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     4081 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     4141 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     4201 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     4261 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     4321 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     4381 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     4441 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4501 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     4561 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     4621 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     4681 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     4741 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     4801 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     4861 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     4921 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     4981 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     5041 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     5101 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     5161 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     5221 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     5281 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     5341 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     5401 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     5461 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     5521 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     5581 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     5641 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     5701 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     5761 atacccacgc cgaaacaa
//
