LOCUS       sequence-245567-        7145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MBP-RGG-GFP-RGG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7145)
  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 7145)
  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-GFP-RGG
FEATURES             Location/Qualifiers
     source          1..7145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     54..73
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     157..176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        157..175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    176..200
                     /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             215..237
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             245..1345
                     /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     1319..1342
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1394..1414
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1928..1945
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(1946..1948,1949..1951,1952..2662)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1946..1948,1949..1951,1952..2662)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1991..2012)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2252..2271)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2599..2620
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2669..2686
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3197..3214
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3267..3285)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3281..3328
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3365..3820
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3452..3471)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3662..3683
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3935..4750)
                     /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(3935..4750)
                     /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     4658..4677
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4742..4761
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4903..5491
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5392..5411
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5645..5662
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(5833..6915)
                     /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     6877..6896
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6916..6993)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
       61 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
      121 ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat
      181 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
      241 acatatgaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
      301 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
      361 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
      421 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
      481 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
      541 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
      601 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
      661 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
      721 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
      781 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
      841 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
      901 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
      961 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
     1021 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcaaaaga
     1081 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
     1141 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gtgaaagatc cgcgtattgc
     1201 cgccactatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
     1261 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga
     1321 tgaagccctg aaagacgcgc agactaattc gagctcgaac aacaacaaca ataacaataa
     1381 caacaacctc ggagagaacc tgtattttca gggtggtatg gaatccaatc aatccaataa
     1441 cggtggctcg ggtaatgccg ctctgaatcg tggtggccgc tacgtgccgc cgcatctgcg
     1501 tggtggcgat ggtggtgcag cagcagctgc atctgctggc ggtgatgacc gtcgcggcgg
     1561 tgcgggcggt ggcggttatc gtcgcggcgg tggcaatagt ggtggcggtg gcggtggcgg
     1621 ttatgatcgt ggttacaatg acaaccgtga tgaccgcgat aaccgtggcg gttccggcgg
     1681 ttacggtcgt gatcgtaatt atgaagaccg tggttacaac ggcggtggcg gtggcggtgg
     1741 caatcgtggc tataacaata accgtggtgg cggtggcggt ggctacaatc gccaggatcg
     1801 tggtgacggt ggcagctcta acttttcgcg cggtggctat aataaccgtg atgaaggcag
     1861 cgacaaccgc ggtagtggcc gttcctacaa taacgatcgt cgcgacaatg gtggcgatgg
     1921 cgaattctta gtacctcgcg ggtcaatggt gagcaagggc gaggagctgt tcaccggggt
     1981 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2041 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2101 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     2161 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2221 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     2281 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2341 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2401 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     2461 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2521 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     2581 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     2641 cggcatggac gagctgtaca aggaattctt agtacctcgc gggtcaatgg aatccaatca
     2701 atccaataac ggtggctcgg gtaatgccgc tctgaatcgt ggtggccgct acgtgccgcc
     2761 gcatctgcgt ggtggcgatg gtggtgcagc agcagctgca tctgctggcg gtgatgaccg
     2821 tcgcggcggt gcgggcggtg gcggttatcg tcgcggcggt ggcaatagtg gtggcggtgg
     2881 cggtggcggt tatgatcgtg gttacaatga caaccgtgat gaccgcgata accgtggcgg
     2941 ttccggcggt tacggtcgtg atcgtaatta tgaagaccgt ggttacaacg gcggtggcgg
     3001 tggcggtggc aatcgtggct ataacaataa ccgtggtggc ggtggcggtg gctacaatcg
     3061 ccaggatcgt ggtgacggtg gcagctctaa cttttcgcgc ggtggctata ataaccgtga
     3121 tgaaggcagc gacaaccgcg gtagtggccg ttcctacaat aacgatcgtc gcgacaatgg
     3181 tggcgatggc ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     3241 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     3301 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     3361 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3421 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3481 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3541 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3601 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3661 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     3721 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3781 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggta
     3841 cccaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3901 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     3961 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     4021 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     4081 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     4141 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     4201 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     4261 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     4321 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     4381 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     4441 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     4501 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     4561 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     4621 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     4681 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     4741 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     4801 accaaaatcc cttaacgtga cctaggtcat gaccaaaatc ccttaacgtg agttttcgtt
     4861 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4921 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4981 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5041 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5101 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5161 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5221 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5281 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5341 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5401 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5461 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5521 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5581 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5641 gcgcagcgag tcagtgagcg aggaagcgga agagcgccat aatccatgat aaagaagaca
     5701 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     5761 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     5821 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5881 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     5941 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     6001 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     6061 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     6121 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     6181 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     6241 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     6301 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     6361 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6421 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6481 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     6541 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     6601 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     6661 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     6721 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     6781 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     6841 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     6901 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     6961 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     7021 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     7081 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     7141 taccc
//
