LOCUS       sequence-245548-        5963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Exoression of RGG-RFP-RGG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5963)
  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 5963)
  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-RFP-RGG
FEATURES             Location/Qualifiers
     source          1..5963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             200..217
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             218..925
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(363..381)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     631..650
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             932..949
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1460..1477
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1530..1548)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1544..1591
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1628..2083
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1715..1734)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1946
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2198..3013)
                     /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(2198..3013)
                     /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     2921..2940
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3005..3024
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3166..3754
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3655..3674
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3908..3925
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(4096..5178)
                     /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     5140..5159
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5179..5256)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5462..5481
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5565..5584
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5565..5583
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5584..5608
                     /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             5623..5645
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 cggtggcggt ggcaatcgtg gctataacaa taaccgtggt ggcggtggcg gtggctacaa
       61 tcgccaggat cgtggtgacg gtggcagctc taacttttcg cgcggtggct ataataaccg
      121 tgatgaaggc agcgacaacc gcggtagtgg ccgttcctac aataacgatc gtcgcgacaa
      181 tggtggcgat ggcgaattct tagtacctcg cgggtcaatg gtgagcaagg gcgaggagga
      241 taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa
      301 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac
      361 cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc
      421 tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt
      481 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
      541 cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa
      601 gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg
      661 ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca
      721 gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc
      781 caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc
      841 ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac
      901 cggcggcatg gacgagctgt acaaggaatt cttagtacct cgcgggtcaa tggaatccaa
      961 tcaatccaat aacggtggct cgggtaatgc cgctctgaat cgtggtggcc gctacgtgcc
     1021 gccgcatctg cgtggtggcg atggtggtgc agcagcagct gcatctgctg gcggtgatga
     1081 ccgtcgcggc ggtgcgggcg gtggcggtta tcgtcgcggc ggtggcaata gtggtggcgg
     1141 tggcggtggc ggttatgatc gtggttacaa tgacaaccgt gatgaccgcg ataaccgtgg
     1201 cggttccggc ggttacggtc gtgatcgtaa ttatgaagac cgtggttaca acggcggtgg
     1261 cggtggcggt ggcaatcgtg gctataacaa taaccgtggt ggcggtggcg gtggctacaa
     1321 tcgccaggat cgtggtgacg gtggcagctc taacttttcg cgcggtggct ataataaccg
     1381 tgatgaaggc agcgacaacc gcggtagtgg ccgttcctac aataacgatc gtcgcgacaa
     1441 tggtggcgat ggcctcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
     1501 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1561 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     1621 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1681 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1741 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1801 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     1861 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     1921 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     1981 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2041 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     2101 gtacccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2161 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     2221 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     2281 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     2341 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     2401 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     2461 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     2521 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     2581 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     2641 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     2701 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     2761 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     2821 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2881 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     2941 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     3001 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     3061 atgaccaaaa tcccttaacg tgacctaggt catgaccaaa atcccttaac gtgagttttc
     3121 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3181 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3241 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3301 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3361 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3421 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3481 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3541 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3601 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3661 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3721 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3781 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3841 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3901 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc cataatccat gataaagaag
     3961 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     4021 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4081 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4141 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4201 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4261 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     4321 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     4381 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     4441 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4501 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4561 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     4621 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     4681 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     4741 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     4801 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     4861 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     4921 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     4981 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     5041 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     5101 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     5161 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     5221 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     5281 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     5341 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     5401 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     5461 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     5521 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     5581 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     5641 ggagatatac atatggaatc caatcaatcc aataacggtg gctcgggtaa tgccgctctg
     5701 aatcgtggtg gccgctacgt gccgccgcat ctgcgtggtg gcgatggtgg tgcagcagca
     5761 gctgcatctg ctggcggtga tgaccgtcgc ggcggtgcgg gcggtggcgg ttatcgtcgc
     5821 ggcggtggca atagtggtgg cggtggcggt ggcggttatg atcgtggtta caatgacaac
     5881 cgtgatgacc gcgataaccg tggcggttcc ggcggttacg gtcgtgatcg taattatgaa
     5941 gaccgtggtt acaacggcgg tgg
//
