LOCUS       Exported                8031 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses MBP-tagged full length FUS.
ACCESSION   .
VERSION     .
KEYWORDS    MBP-FUS_FL_WT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8031)
  AUTHORS   Burke KA, Janke AM, Rhine CL, Fawzi NL
  TITLE     Residue-by-Residue View of In Vitro FUS Granules that Bind the 
            C-Terminal Domain of RNA Polymerase II.
  JOURNAL   Mol Cell. 2015 Oct 15;60(2):231-41. doi: 
            10.1016/j.molcel.2015.09.006. Epub 2015 Oct 8.
  PUBMED    26455390
REFERENCE   2  (bases 1 to 8031)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8031
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             106..123
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             136..1233
                     /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"
     regulatory      1090..1099
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     1207..1230
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1252..1276
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1285..1305
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1689..1700
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             2896..2913
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2966..2984)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2980..3027
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3064..3519
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3151..3170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3361..3382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3611..4426)
                     /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     4334..4353
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4418..4437
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4548..5136
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5037..5056
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5290..5307
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5322..5461
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5387..5406)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5547..5569
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5563..5754)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6563..7645)
                     /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     7607..7626
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7646..7723)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7929..7948
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggatctgata aaattcatca tcatcatcat
      121 cacagcagcg gtaccaaaat cgaagaaggt aaactggtaa tctggattaa cggcgataaa
      181 ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc
      241 accgttgagc atccggataa actggaagag aaattcccac aggttgcggc aactggcgat
      301 ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg
      361 ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat
      421 gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg
      481 atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg
      541 gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac
      601 ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag
      661 tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg
      721 gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct
      781 gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc
      841 gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc
      901 aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg
      961 gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa
     1021 gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggc gaaagatcca
     1081 cgtattgccg ccaccatgga aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag
     1141 atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag
     1201 actgtcgatg aagccctgaa agacgcgcag actaattcgg gatctgatat cacaagtttg
     1261 tacaaaaaag ctgaaggcgg taccgagaac ctgtacttcc agggtcatat ggcctcaaac
     1321 gattataccc aacaagcaac ccaaagctat ggggcctacc ccacccagcc cgggcagggc
     1381 tattcccagc agagcagtca gccctacgga cagcagagtt acagtggtta tagccagtcc
     1441 acggacactt caggctatgg ccagagcagc tattcttctt atggccagag ccagaacaca
     1501 ggctatggaa ctcagtcaac tccccaggga tatggctcga ctggcggcta tggtagtagc
     1561 cagagctccc aatcgtctta cgggcagcag tcctcctatc ctggctatgg ccagcagcca
     1621 gctcccagca gcacctcggg aagttacggt agcagttctc agagcagcag ctatgggcag
     1681 ccccagagtg ggagctacag ccagcagcct agctatggtg gacagcagca aagctatgga
     1741 cagcagcaaa gctataatcc ccctcagggc tatggacagc agaaccagta caacagcagc
     1801 agtggtggtg gaggtggagg tggaggtgga ggtaactatg gccaagatca atcctccatg
     1861 agtagtggtg gtggcagtgg tggcggttat ggcaatcaag accagagtgg tggaggtggc
     1921 agcggtggct atggacagca ggaccgtgga ggccgcggca ggggtggcag tggtggcggc
     1981 ggcggcggcg gcggtggtgg ttacaaccgc agcagtggtg gctatgaacc cagaggtcgt
     2041 ggaggtggcc gtggaggcag aggtggcatg ggcggaagtg accgtggtgg cttcaataaa
     2101 tttggtggcc ctcgggacca aggatcacgt catgactccg aacaggataa ttcagacaac
     2161 aacaccatct ttgtgcaagg cctgggtgag aatgttacaa ttgagtctgt ggctgattac
     2221 ttcaagcaga ttggtattat taagacaaac aagaaaacgg gacagcccat gattaatttg
     2281 tacacagaca gggaaactgg caagctgaag ggagaggcaa cggtctcttt tgatgaccca
     2341 ccttcagcta aagcagctat tgactggttt gatggtaaag aattctccgg aaatcctatc
     2401 aaggtctcat ttgctactcg ccgggcagac tttaatcggg gtggtggcaa tggtcgtgga
     2461 ggccgagggc gaggaggacc catgggccgt ggaggctatg gaggtggtgg cagtggtggt
     2521 ggtggccgag gaggatttcc cagtggaggt ggtggcggtg gaggacagca gcgagctggt
     2581 gactggaagt gtcctaatcc cacctgtgag aatatgaact tctcttggag gaatgaatgc
     2641 aaccagtgta aggcccctaa accagatggc ccaggagggg gaccaggtgg ctctcacatg
     2701 gggggtaact acggggatga tcgtcgtggt ggcagaggag gctatgatcg aggcggctac
     2761 cggggccgcg gcggggaccg tggaggcttc cgagggggcc ggggtggtgg ggacagaggt
     2821 ggctttggcc ctggcaagat ggattccagg ggtgagcaca gacaggatcg cagggagagg
     2881 ccgtattgac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     2941 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     3001 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     3061 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3121 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3181 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3241 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3301 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3361 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3421 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3481 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     3541 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3601 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     3661 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     3721 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     3781 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     3841 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     3901 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     3961 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     4021 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     4081 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     4141 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     4201 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     4261 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     4321 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     4381 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     4441 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     4501 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg atatcctttt
     4561 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4621 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4681 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4741 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4801 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4861 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4921 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4981 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5041 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5101 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5161 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5221 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5281 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     5341 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     5401 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     5461 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5521 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5581 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     5641 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     5701 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     5761 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     5821 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     5881 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     5941 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     6001 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     6061 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     6121 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     6181 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     6241 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     6301 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     6361 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     6421 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     6481 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     6541 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6601 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     6661 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     6721 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     6781 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     6841 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     6901 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     6961 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     7021 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     7081 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     7141 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     7201 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     7261 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     7321 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     7381 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     7441 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     7501 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     7561 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     7621 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     7681 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     7741 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     7801 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     7861 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     7921 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     7981 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t
//
