LOCUS       sequence-485123-        6638 bp    DNA     circular SYN 02-SEP-2025
DEFINITION  Expresses the MBP-GFP-RGG fusion protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6638)
  AUTHORS   Kelley FM, Favetta B, Regy RM, Mittal J, Schuster BS
  TITLE     Amphiphilic proteins coassemble into multiphasic condensates and act
            as biomolecular surfactants.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Dec 21;118(51):e2109967118. doi: 
            10.1073/pnas.2109967118.
  PUBMED    34916288
REFERENCE   2  (bases 1 to 6638)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2021-12-21"; volume: "118(51):e2109967118. 
            doi"; pages: " 10.1073/pnas.2109967118"
COMMENT     Sequence Label: MBP-GFP-RGG
FEATURES             Location/Qualifiers
     source          1..6638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     309..330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(582..1397)
                     /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(582..1397)
                     /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     1305..1324
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1389..1408
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1550..2138
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2039..2058
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2292..2309
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(2480..3562)
                     /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     3524..3543
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3563..3640)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3846..3865
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3949..3968
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3949..3967
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3968..3992
                     /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             4007..4029
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4037..5137
                     /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     5111..5134
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5186..5206
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site
                     "
                     /label=TVMV site
                     /translation="ETVRFQS"
     CDS             join(5207..5209,5210..5212,5213..5923)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5252..5273)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5513..5532)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5860..5881
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5936..5956
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             6482..6499
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6552..6570)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6566..6613
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggtaccc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      541 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
      601 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
      661 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
      721 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
      781 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
      841 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
      901 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
      961 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     1021 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     1081 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     1141 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     1201 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     1261 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     1321 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     1381 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     1441 gttcatgacc aaaatccctt aacgtgacct aggtcatgac caaaatccct taacgtgagt
     1501 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1561 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1621 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1681 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1741 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     1801 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     1861 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     1921 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     1981 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2041 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2101 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2161 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2221 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2281 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgccataat ccatgataaa
     2341 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     2401 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     2461 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     2521 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     2581 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     2641 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     2701 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     2761 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     2821 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     2881 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     2941 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     3001 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     3061 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     3121 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     3181 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     3241 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     3301 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     3361 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     3421 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     3481 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     3541 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     3601 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     3661 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     3721 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     3781 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     3841 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     3901 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     3961 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     4021 agaaggagat atacatatga aaatcgaaga aggtaaactg gtaatctgga ttaacggcga
     4081 taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata ccggaattaa
     4141 agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg cggcaactgg
     4201 cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg
     4261 cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc cgtttacctg
     4321 ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg aagcgttatc
     4381 gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag agatcccggc
     4441 gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc
     4501 gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt atgaaaacgg
     4561 caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt
     4621 cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact ccatcgcaga
     4681 agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg catggtccaa
     4741 catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca agggtcaacc
     4801 atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc cgaacaaaga
     4861 gctggcaaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg aagcggttaa
     4921 taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt tggtgaaaga
     4981 tccgcgtatt gccgccacta tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc
     5041 gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg
     5101 tcagactgtc gatgaagccc tgaaagacgc gcagactaat tcgagctcga acaacaacaa
     5161 caataacaat aacaacaacc tcggagagac cgtgcgtttc cagtctatgg tgagcaaggg
     5221 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     5281 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     5341 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     5401 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     5461 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     5521 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     5581 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     5641 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     5701 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     5761 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     5821 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     5881 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggcg gatctgagaa
     5941 tttatatttc cagggcgaat tcgggaagct tatggaatcc aatcaatcca ataacggtgg
     6001 ctcgggtaat gccgctctga atcgtggtgg ccgctacgtg ccgccgcatc tgcgtggtgg
     6061 cgatggtggt gcagcagcag ctgcatctgc tggcggtgat gaccgtcgcg gcggtgcggg
     6121 cggtggcggt tatcgtcgcg gcggtggcaa tagtggtggc ggtggcggtg gcggttatga
     6181 tcgtggttac aatgacaacc gtgatgaccg cgataaccgt ggcggttccg gcggttacgg
     6241 tcgtgatcgt aattatgaag accgtggtta caacggcggt ggcggtggcg gtggcaatcg
     6301 tggctataac aataaccgtg gtggcggtgg cggtggctac aatcgccagg atcgtggtga
     6361 cggtggcagc tctaactttt cgcgcggtgg ctataataac cgtgatgaag gcagcgacaa
     6421 ccgcggtagt ggccgttcct acaataacga tcgtcgcgac aatggtggcg atggcctcga
     6481 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     6541 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     6601 gaggggtttt ttgctgaaag gaggaactat atccggat
//
