LOCUS       sequence-454672-        6309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (S2-S4)x1, with short 
            flexible linkers and N-terminal TagBFP; 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 6309)
  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 6309)
  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: TagBFP(S2-S4)1-gs
FEATURES             Location/Qualifiers
     source          1..6309
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             953..970
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             971..1669
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     promoter        complement(1862..1880)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1863..1880)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1900..1917)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1906..2130
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2176..2604
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2263..2282)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2473..2494
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2613..2633)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2618..2947
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2798..2933
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2860..2879
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3014..3808
                     /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(3068..3087)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3678..3697
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3982..4103
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4019..4038)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4073..4092
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4152..4168)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4152..4168)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4165..4187)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4176..4192
                     /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)."
     promoter        complement(join(4200..4206,4207..4224,4225..4230))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4245..4266
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4383..4400)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4554..5142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4634..4653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5313..6104,6105..6173))
                     /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     5936..5955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6174..6278)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcgccacca tgcaccacca
      961 ccaccaccac atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     1021 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     1081 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     1141 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
     1201 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     1261 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     1321 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     1381 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
     1441 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     1501 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     1561 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     1621 agtggccaga tactgcgacc tccctagcaa actggggcac aagctcaatg gcagtggctc
     1681 cccagggagc ggtgagatac aaagtcttga ggagaagata agtcagctta agcaggagaa
     1741 ctctcaattg gaggaaggtt ccgggagtcc gggaagtgga aaaatccaga gtttgaaaga
     1801 aaagaactcc caactcaaac aagagatctc tcaactggaa gaataatcta gagggcccta
     1861 ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt
     1921 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     1981 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2041 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2101 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2161 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2221 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2281 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2341 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2401 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2461 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2521 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2581 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     2641 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2701 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2761 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     2821 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     2881 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     2941 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     3001 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3061 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3121 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3181 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3241 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     3301 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     3361 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     3421 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     3481 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     3541 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     3601 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     3661 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     3721 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     3781 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     3841 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     3901 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     3961 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4021 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4081 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4141 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4201 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4261 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4321 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4381 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4441 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4501 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4561 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4621 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4681 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4741 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4801 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4861 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4921 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4981 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5041 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5101 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5161 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5221 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5281 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5341 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5401 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5461 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5521 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5581 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5641 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5701 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5761 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5821 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5881 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5941 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6001 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6061 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6121 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6181 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6241 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6301 cctgacgtc
//
