LOCUS       sequence-451390-        6966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of 10 coiled coil segments in PPPPPPAAAA 
            arrangement, with short flexible linkers and N-terminal mCitrine for
            phase separation and forming of liquid condensates in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6966)
  AUTHORS   Ramsak M, Ramirez DA, Hough LE, Shirts MR, Vidmar S, Elersic Filipic
            K, Anderluh G, Jerala R
  TITLE     Programmable de novo designed coiled coil-mediated phase separation 
            in mammalian cells.
  JOURNAL   Nat Commun. 2023 Dec 2;14(1):7973. doi: 10.1038/s41467-023-43742-w.
  PUBMED    38042897
REFERENCE   2  (bases 1 to 6966)
  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: "2023-12-2"; volume: "14(1):7973. doi"; pages: " 
            10.1038/s41467-023-43742-w"
COMMENT     Sequence Label: RFP-PPPPPPAAAA-gs
FEATURES             Location/Qualifiers
     source          1..6966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     172..189
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        209..227
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     209..226
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(600..656)
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             complement(684..740)
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             complement(768..824)
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             complement(852..908)
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             complement(936..992)
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             complement(1020..1076)
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             complement(1104..1775)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             complement(1776..1793)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1864..1883)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1865..1883)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1928..2131)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1957..1977)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2132..2511
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2683..2702
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2777..2881
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2882..2950,2951..3742)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3100..3119)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3913..4501
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4402..4421
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4655..4672
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4789..4810
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4825..4830,4831..4848,4849..4855)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4863..4879
                     /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)."
     primer_bind     4868..4890
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4887..4903
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4887..4903
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4952..5073
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4963..4982)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5017..5036
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5247..6041)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5358..5377)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5968..5987
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(6108..6437)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6122..6257
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6176..6195)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     6422..6442
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(6451..6879)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6561..6582)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6773..6792
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    join(6925..6966,1..183)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac
       61 tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt
      121 ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca
      181 cagtcgaggc tgatcagcga gctctagcat ttaggtgaca ctatagaata gggccctcta
      241 gattactcct tcctggcttg tgctttccac tgcagctgtg cgagttgctc ctctattgct
      301 tgcagttcgg aacccccgct accacttccc tcttttcgcg cttgtgcctt ccattgcaac
      361 tgggcgagtt gttcttcgat ggcctgaagt tctgagccgc cacttcctga cccttccttg
      421 cgagcttgag ctttccattg gagttgtgcc aattgttctt ctattgcctg caattcacta
      481 ccaccggaac cactaccttc ttttctggcc tgcgctttcc attgcagttg cgccaactgc
      541 tcttctatag cttggagctc agagcctccg gagcctccgg agccgccgcc agagccgccc
      601 ttcttcagcc gtgccacttc gttttccaag tgataattct tagacaacaa ttcttctact
      661 ttggatcccc cagatcctga ccccttctta agtcgggcta cctcgttctc caaatggtaa
      721 ttcttactca gaagttcctc tactttagac ccgccggatc cggacccttt tttcagccta
      781 gcgacttcat tttcaagatg atagttcttg ctgagaagtt cctccacctt ggagccccct
      841 gagccactgc cttttttcaa tcgtgcgact tcgttctcga ggtgataatt tttagaaagc
      901 aattcttcga ctttagaccc accggaaccg cttcccttct tcaaccgggc gacttcgttt
      961 tcgagatggt agtttttact gagaagttct tccactttgc tacctccaga acctgatcct
     1021 ttcttcagtc tagctacctc attttcaagg tgataattct tactcaagag ctcctcgacc
     1081 ttgctcccac cagagcctga gccagcaccg gtggagtgac gaccttcagc acgttcgtac
     1141 tgttcaacga tggtgtagtc ttcgttgtgg gaggtgatgt ccagtttgat gtcggttttg
     1201 taagcacccg gcagctgaac cggtttttta gccatgtagg tggttttaac ttcagcgtcg
     1261 tagtgaccac cgtctttcag tttcagacgc attttgattt cacctttcag agcaccgtct
     1321 tccgggtaca tacgttcggt ggaagcttcc caacccatgg tttttttctg cataaccgga
     1381 ccgtcggacg ggaagttggt accacgcagt ttaactttgt agatgaactc accgtcttgc
     1441 agggaggagt cctgggtaac ggtaacaaca ccaccgtctt cgaagttcat aacacgttcc
     1501 catttgaaac cttccgggaa ggacagtttc aggtagtccg ggatgtcagc cgggtgttta
     1561 acgtaagctt tggaaccgta ctggaactgc ggggacagga tgtcccaagc gaacggcagc
     1621 ggaccacctt tggtaacttt cagtttagcg gtctgggtac cttcgtacgg acgaccttca
     1681 ccttcacctt cgatttcgaa ctcgtgaccg ttaacggaac cttccatacg aactttgaaa
     1741 cgcatgaact ctttgataac gtcttcggag gaagcgtggt ggtggtggtg gtgcatggtg
     1801 gcgaattcca gcacactggc ggccgttact agtggatccg agctcggtac caagcttggg
     1861 tctccctata gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta
     1921 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     1981 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     2041 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     2101 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     2161 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     2221 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     2281 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     2341 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     2401 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     2461 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat
     2521 atctggcccg tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc
     2581 aattgtcggt caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga
     2641 cctccaacac acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg
     2701 tactgagagt gcaccatagg ggatcgggag atctcccgat ccgtcgacgt caggtggcac
     2761 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2821 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2881 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2941 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3001 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3061 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3121 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3181 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3241 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3301 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3361 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3421 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3481 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3541 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3601 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3661 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     3721 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3781 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3841 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3901 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3961 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4021 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4081 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4141 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4201 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4261 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4321 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4381 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4441 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4501 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4561 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4621 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4681 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     4741 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     4801 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     4861 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     4921 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     4981 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     5041 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     5101 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     5161 ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc
     5221 ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     5281 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     5341 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     5401 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     5461 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     5521 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     5581 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     5641 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     5701 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     5761 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     5821 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     5881 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     5941 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     6001 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     6061 gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct
     6121 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg
     6181 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     6241 ggagttaggg gcgggatggg cggagttagg ggcgggrcta tggttgctga ctaattgaga
     6301 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     6361 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     6421 caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct
     6481 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     6541 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     6601 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     6661 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     6721 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     6781 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     6841 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc
     6901 agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
     6961 tgtctt
//
