LOCUS       sequence-246036-        5782 bp    DNA     circular SYN 14-APR-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 Apr 14, 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
FEATURES             Location/Qualifiers
     source          1..5782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..119
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     203..222
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        203..221
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    222..246
                     /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             261..283
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             326..346
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             727..738
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             849..866
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(919..937)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      933..980
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1017..1472
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1104..1123)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1314..1335
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1564..2379)
                     /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(1564..2379)
                     /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     2287..2306
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2371..2390
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2501..3089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2990..3009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3243..3260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3275..3414
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3340..3359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3500..3522
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3516..3707)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4516..5598)
                     /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     5560..5579
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5599..5676)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
       61 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
      121 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
      181 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
      241 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tgggctctga
      301 taaaattcat catcatcatc atcacgaaaa tctttatttt caaggtatgg cctcaaacga
      361 ttatacccaa caagcaaccc aaagctatgg ggcctacccc acccagcccg ggcagggcta
      421 ttcccagcag agcagtcagc cctacggaca gcagagttac agtggttata gccagtccac
      481 ggacacttca ggctatggcc agagcagcta ttcttcttat ggccagagcc agaacacagg
      541 ctatggaact cagtcaactc cccagggata tggctcgact ggcggctatg gcagtagcca
      601 gagctcccaa tcgtcttacg ggcagcagtc ctcctatcct ggctatggcc agcagccagc
      661 tcccagcagc acctcgggaa gttacggtag cagttctcag agcagcagct atgggcagcc
      721 ccagagtggg agctacagcc agcagcctag ctatggtgga cagcagcaaa gctatggaca
      781 gcagcaaagc tataatcccc ctcagggcta tggacagcag aaccagtaca acagctgaga
      841 tcctcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
      901 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
      961 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1021 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1081 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1141 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1201 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1261 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1321 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1381 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1441 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     1501 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     1561 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     1621 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     1681 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     1741 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     1801 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     1861 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     1921 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     1981 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     2041 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     2101 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     2161 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     2221 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     2281 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     2341 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     2401 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2461 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2521 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2581 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2641 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2701 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2761 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2821 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2881 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2941 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3001 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3061 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3121 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3181 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3241 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3301 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     3361 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3421 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3481 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3541 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3601 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3661 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3721 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     3781 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     3841 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     3901 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     3961 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4021 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4081 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4141 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     4201 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     4261 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     4321 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     4381 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     4441 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4501 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4561 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4621 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4681 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     4741 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     4801 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     4861 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4921 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4981 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5041 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     5101 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     5161 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     5221 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     5281 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     5341 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     5401 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     5461 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     5521 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     5581 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     5641 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     5701 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     5761 caaggaatgg tgcatgcaag ga
//
