LOCUS       sequence-245565-        5972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RGG-GFP-RGG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5972)
  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 5972)
  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_RGG-GFP-RGG
FEATURES             Location/Qualifiers
     source          1..5972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..48
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     132..151
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        132..150
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    151..175
                     /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             190..212
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             730..747
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(748..750,751..753,754..1464)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(748..750,751..753,754..1464)
                     /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(793..814)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1054..1073)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1401..1422
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1471..1488
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1999..2016
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2069..2087)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2083..2130
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2167..2622
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2254..2273)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2464..2485
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2737..3552)
                     /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(2737..3552)
                     /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     3460..3479
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3544..3563
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3705..4293
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4194..4213
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4447..4464
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(4635..5717)
                     /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     5679..5698
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5718..5795)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
       61 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
      121 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
      181 agaaataatt ttgtttaact ttaagaagga gatatacata tggaatccaa tcaatccaat
      241 aacggtggct cgggtaatgc cgctctgaat cgtggtggcc gctacgtgcc gccgcatctg
      301 cgtggtggcg atggtggtgc agcagcagct gcatctgctg gcggtgatga ccgtcgcggc
      361 ggtgcgggcg gtggcggtta tcgtcgcggc ggtggcaata gtggtggcgg tggcggtggc
      421 ggttatgatc gtggttacaa tgacaaccgt gatgaccgcg ataaccgtgg cggttccggc
      481 ggttacggtc gtgatcgtaa ttatgaagac cgtggttaca acggcggtgg cggtggcggt
      541 ggcaatcgtg gctataacaa taaccgtggt ggcggtggcg gtggctacaa tcgccaggat
      601 cgtggtgacg gtggcagctc taacttttcg cgcggtggct ataataaccg tgatgaaggc
      661 agcgacaacc gcggtagtgg ccgttcctac aataacgatc gtcgcgacaa tggtggcgat
      721 ggcgaattct tagtacctcg cgggtcaatg gtgagcaagg gcgaggagct gttcaccggg
      781 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
      841 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
      901 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
      961 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1021 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1081 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1141 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1201 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1261 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1321 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     1381 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     1441 ctcggcatgg acgagctgta caaggaattc ttagtacctc gcgggtcaat ggaatccaat
     1501 caatccaata acggtggctc gggtaatgcc gctctgaatc gtggtggccg ctacgtgccg
     1561 ccgcatctgc gtggtggcga tggtggtgca gcagcagctg catctgctgg cggtgatgac
     1621 cgtcgcggcg gtgcgggcgg tggcggttat cgtcgcggcg gtggcaatag tggtggcggt
     1681 ggcggtggcg gttatgatcg tggttacaat gacaaccgtg atgaccgcga taaccgtggc
     1741 ggttccggcg gttacggtcg tgatcgtaat tatgaagacc gtggttacaa cggcggtggc
     1801 ggtggcggtg gcaatcgtgg ctataacaat aaccgtggtg gcggtggcgg tggctacaat
     1861 cgccaggatc gtggtgacgg tggcagctct aacttttcgc gcggtggcta taataaccgt
     1921 gatgaaggca gcgacaaccg cggtagtggc cgttcctaca ataacgatcg tcgcgacaat
     1981 ggtggcgatg gcctcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     2041 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     2101 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     2161 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2221 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2281 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2341 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2401 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2461 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2521 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2581 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     2641 tacccaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2701 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     2761 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     2821 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     2881 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     2941 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     3001 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     3061 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     3121 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     3181 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     3241 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     3301 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     3361 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     3421 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     3481 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     3541 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     3601 tgaccaaaat cccttaacgt gacctaggtc atgaccaaaa tcccttaacg tgagttttcg
     3661 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3721 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3781 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3841 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3901 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3961 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4021 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4081 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4141 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4201 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4261 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4321 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4381 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4441 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc ataatccatg ataaagaaga
     4501 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     4561 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     4621 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4681 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     4741 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     4801 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4861 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     4921 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4981 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     5041 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     5101 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     5161 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     5221 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     5281 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     5341 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     5401 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     5461 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     5521 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     5581 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     5641 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     5701 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     5761 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     5821 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     5881 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     5941 catacccacg ccgaaacaag cgctcatgag cc
//
