LOCUS       sequence-245556-        6422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MBP-RGG-RGG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6422)
  AUTHORS   Schuster BS, Reed EH, Parthasarathy R, Jahnke CN, Caldwell RM, 
            Bermudez JG, Ramage H, Good MC, Hammer DA
  TITLE     Controllable protein phase separation and modular recruitment to 
            form responsive membraneless organelles.
  JOURNAL   Nat Commun. 2018 Jul 30;9(1):2985. doi: 10.1038/s41467-018-05403-1.
  PUBMED    30061688
REFERENCE   2  (bases 1 to 6422)
  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"; journalName: "Nat 
            Commun."; date: "2018-07-30"; volume: "9(1):2985. doi"; pages: " 
            10.1038/s41467-018-05403-1"
COMMENT     Sequence Label: pET_MBP-RGG-RGG
FEATURES             Location/Qualifiers
     source          1..6422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..62
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     146..165
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        146..164
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    165..189
                     /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             204..226
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             237..254
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     regulatory      251..260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             267..1367
                     /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="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1341..1364
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1398..1421
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1935..1952
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2463..2480
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2533..2551)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2547..2594
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2631..3086
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2718..2737)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2928..2949
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3201..4016)
                     /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(3201..4016)
                     /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     3924..3943
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4008..4027
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4169..4757
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4658..4677
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4911..4928
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(5099..6181)
                     /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     6143..6162
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6182..6259)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
       61 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
      121 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
      181 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatgcacc
      241 atcaccatca ccatggcggc ggatctatga aaatcgaaga aggtaaactg gtaatctgga
      301 ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata
      361 ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg
      421 cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg
      481 ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc
      541 cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg
      601 aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag
      661 agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc
      721 tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt
      781 atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg
      841 gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact
      901 ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg
      961 catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca
     1021 agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc
     1081 cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg
     1141 aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt
     1201 tggtgaaaga tccgcgtatt gccgccacta tggaaaacgc ccagaaaggt gaaatcatgc
     1261 cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg
     1321 ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagaccggt gggggaggga
     1381 gtggcggtgg tggcagcctt gaagtcctct ttcaggggcc gggtatggaa tccaatcaat
     1441 ccaataacgg tggctcgggt aatgccgctc tgaatcgtgg tggccgctac gtgccgccgc
     1501 atctgcgtgg tggcgatggt ggtgcagcag cagctgcatc tgctggcggt gatgaccgtc
     1561 gcggcggtgc gggcggtggc ggttatcgtc gcggcggtgg caatagtggt ggcggtggcg
     1621 gtggcggtta tgatcgtggt tacaatgaca accgtgatga ccgcgataac cgtggcggtt
     1681 ccggcggtta cggtcgtgat cgtaattatg aagaccgtgg ttacaacggc ggtggcggtg
     1741 gcggtggcaa tcgtggctat aacaataacc gtggtggcgg tggcggtggc tacaatcgcc
     1801 aggatcgtgg tgacggtggc agctctaact tttcgcgcgg tggctataat aaccgtgatg
     1861 aaggcagcga caaccgcggt agtggccgtt cctacaataa cgatcgtcgc gacaatggtg
     1921 gcgatggcga attcttagta cctcgcgggt caatggaatc caatcaatcc aataacggtg
     1981 gctcgggtaa tgccgctctg aatcgtggtg gccgctacgt gccgccgcat ctgcgtggtg
     2041 gcgatggtgg tgcagcagca gctgcatctg ctggcggtga tgaccgtcgc ggcggtgcgg
     2101 gcggtggcgg ttatcgtcgc ggcggtggca atagtggtgg cggtggcggt ggcggttatg
     2161 atcgtggtta caatgacaac cgtgatgacc gcgataaccg tggcggttcc ggcggttacg
     2221 gtcgtgatcg taattatgaa gaccgtggtt acaacggcgg tggcggtggc ggtggcaatc
     2281 gtggctataa caataaccgt ggtggcggtg gcggtggcta caatcgccag gatcgtggtg
     2341 acggtggcag ctctaacttt tcgcgcggtg gctataataa ccgtgatgaa ggcagcgaca
     2401 accgcggtag tggccgttcc tacaataacg atcgtcgcga caatggtggc gatggcctcg
     2461 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     2521 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     2581 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     2641 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2701 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2761 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2821 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2881 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2941 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3001 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3061 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggtaccca ggtggcactt
     3121 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3181 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     3241 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     3301 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     3361 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     3421 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     3481 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     3541 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     3601 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     3661 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     3721 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     3781 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     3841 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     3901 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     3961 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     4021 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     4081 acgtgaccta ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4141 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4201 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4261 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     4321 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4381 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4441 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4501 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4561 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4621 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4681 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4741 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4801 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4861 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4921 tgagcgagga agcggaagag cgccataatc catgataaag aagacagtca taagtgcggc
     4981 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5041 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5101 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5161 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     5221 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     5281 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     5341 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     5401 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     5461 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     5521 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     5581 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     5641 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     5701 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     5761 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     5821 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     5881 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     5941 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6001 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6061 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6121 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6181 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     6241 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     6301 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     6361 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     6421 ca
//
