LOCUS       sequence-485122-        6146 bp    DNA     circular SYN 02-SEP-2025
DEFINITION  Expresses the GST-GFP-RGG fusion protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6146)
  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 6146)
  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: GST-GFP-RGG
FEATURES             Location/Qualifiers
     source          1..6146
                     /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..4687
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPP"
     primer_bind     4648..4670
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             4694..4714
                     /codon_start=1
                     /product="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site
                     "
                     /label=TVMV site
                     /translation="ETVRFQS"
     CDS             join(4715..4717,4718..4720,4721..5431)
                     /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(4760..4781)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5021..5040)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5368..5389
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5444..5464
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5990..6007
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6060..6078)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6074..6121
                     /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 atacatatgt cccctatact aggttattgg aaaattaagg gccttgtgca
     4081 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
     4141 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
     4201 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
     4261 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
     4321 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
     4381 tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga
     4441 tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt
     4501 gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa
     4561 attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc
     4621 cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca
     4681 tcctccagga tccgaaaccg tgcgtttcca gtctatggtg agcaagggcg aggagctgtt
     4741 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     4801 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     4861 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     4921 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     4981 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     5041 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     5101 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     5161 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     5221 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     5281 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaagctgag
     5341 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     5401 gatcactctc ggcatggacg agctgtacaa gggcggcgga tctgagaatt tatatttcca
     5461 gggcgaattc gggaagctta tggaatccaa tcaatccaat aacggtggct cgggtaatgc
     5521 cgctctgaat cgtggtggcc gctacgtgcc gccgcatctg cgtggtggcg atggtggtgc
     5581 agcagcagct gcatctgctg gcggtgatga ccgtcgcggc ggtgcgggcg gtggcggtta
     5641 tcgtcgcggc ggtggcaata gtggtggcgg tggcggtggc ggttatgatc gtggttacaa
     5701 tgacaaccgt gatgaccgcg ataaccgtgg cggttccggc ggttacggtc gtgatcgtaa
     5761 ttatgaagac cgtggttaca acggcggtgg cggtggcggt ggcaatcgtg gctataacaa
     5821 taaccgtggt ggcggtggcg gtggctacaa tcgccaggat cgtggtgacg gtggcagctc
     5881 taacttttcg cgcggtggct ataataaccg tgatgaaggc agcgacaacc gcggtagtgg
     5941 ccgttcctac aataacgatc gtcgcgacaa tggtggcgat ggcctcgagc accaccacca
     6001 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     6061 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     6121 gctgaaagga ggaactatat ccggat
//
