LOCUS       sequence-451136-        6657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (S2-S4)x3, 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 6657)
  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 6657)
  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)3-gs
FEATURES             Location/Qualifiers
     source          1..6657
                     /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(2210..2228)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2211..2228)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2248..2265)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2254..2478
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2524..2952
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2611..2630)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2821..2842
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2961..2981)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2966..3295
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3146..3281
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3208..3227
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3362..4156
                     /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(3416..3435)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4026..4045
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4330..4451
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4367..4386)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4421..4440
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4500..4516)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4500..4516)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4513..4535)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4524..4540
                     /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(4548..4554,4555..4572,4573..4578))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4593..4614
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4731..4748)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4902..5490)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4982..5001)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5661..6452,6453..6521))
                     /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     6284..6303
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6522..6626)
                     /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 gctccgggag
     1681 cccggggtct ggggaaatac agagcttgga ggagaaaata agtcagctca aacaagagaa
     1741 tagccagctc gaagaaggat ctggtagtcc tggatccggc aagatccaat ctctcaagga
     1801 aaagaattca caactgaagc aggagatttc tcaactggaa gaaggctcag gctcacccgg
     1861 gtccggagag attcaatcac tcgaagaaaa aatatcccaa ctcaaacagg agaacagtca
     1921 gctcgaggag ggatccggct cccctggctc tggaaaaatc caatcattga aggagaagaa
     1981 ctcacagctc aaacaagaaa tttctcagtt ggaggaaggg tcaggaagtc ccggtagcgg
     2041 ggagatccaa tctctggaag agaaaataag tcaactcaag caagaaaaca gtcaacttga
     2101 agaaggtagt ggatccccag gctctggaaa aatacaaagc ctcaaagaaa agaattctca
     2161 actcaaacaa gagattagtc agctggagga gtaatctaga gggccctatt ctatagtgtc
     2221 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2281 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2341 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2401 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2461 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2521 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2581 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2641 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2701 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2761 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2821 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2881 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2941 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3001 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3061 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3121 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3181 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3241 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3301 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     3361 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3421 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3481 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3541 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3601 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3661 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3721 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3781 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3841 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     3901 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3961 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4021 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4081 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4141 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4201 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4261 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4321 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4381 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4441 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4501 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4561 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4621 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4681 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4741 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4801 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4861 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4921 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4981 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5041 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5101 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5161 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5221 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5281 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5341 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5401 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5461 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tactgggtct
     5521 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5581 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5641 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5701 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5761 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5821 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5881 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5941 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6001 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6061 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6121 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6181 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6241 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6301 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6361 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6421 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6481 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6541 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6601 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
