LOCUS       sequence-454671-        7161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (S2)x3-(S4)x3, with 
            long rigid 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 7161)
  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 7161)
  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)3(S4)3-SAH
FEATURES             Location/Qualifiers
     source          1..7161
                     /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
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLIA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     promoter        complement(2714..2732)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2715..2732)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2752..2769)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2758..2982
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3028..3456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3115..3134)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3325..3346
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3465..3485)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3470..3799
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3650..3785
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3712..3731
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3866..4660
                     /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(3920..3939)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4530..4549
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4834..4955
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4871..4890)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4925..4944
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5004..5020)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5004..5020)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5017..5039)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5028..5044
                     /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(5052..5058,5059..5076,5077..5082))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5097..5118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5235..5252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5406..5994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5486..5505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6165..6956,6957..7025))
                     /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     6788..6807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7026..7130)
                     /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 gtgctgatcg 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 aagaagagga
     1681 gaagaaaaag aaagaggagg aagagaaaaa gaaaaaggaa gaagaagaaa agaaaaagaa
     1741 agaagaggag gagaagaaaa aaaaggagga ggaggaagaa attcagtcac ttgaggaaaa
     1801 aatttcacag ttgaagcaag aaaactctca gctggaagaa gaggaagagg aaaagaagaa
     1861 aaaagaagaa gaggagaaga aaaagaagga ggaagaagag aaaaaaaaga aggaagaaga
     1921 agagaaaaag aagaaggagg aagaggaaga gatacaaagt ctggaggaaa aaatatctca
     1981 actgaagcaa gagaacagcc aactggaaga ggaagaggaa gaaaaaaaaa agaaggagga
     2041 agaagaaaag aagaaaaaag aggaagagga aaagaagaag aaagaagaag aggagaaaaa
     2101 gaagaaagag gaggaggaag agattcaatc cctggaggaa aaaatttctc aattgaaaca
     2161 ggagaactca caactggaag aggaagaaga agaaaagaaa aagaaggaag aggaagaaaa
     2221 aaaaaaaaag gaagaagaag aaaagaagaa gaaagaggaa gaagaaaaga agaagaagga
     2281 agaagaggag aaaattcaaa gtctcaaaga aaagaacagc caattgaaac aagaaatctc
     2341 tcagcttgaa gaagaagagg aagaaaagaa aaaaaaggaa gaagaggaaa agaaaaagaa
     2401 agaagaggaa gaaaagaaaa agaaagagga ggaagagaag aaaaaaaaag aggaagaaga
     2461 gaagatacag tcacttaaag aaaagaatag tcagctgaag caggagataa gccagttgga
     2521 ggaggaagaa gaagagaaga agaaaaagga agaggaggaa aagaaaaaga aggaggaaga
     2581 ggagaagaag aagaaggaag aagaagagaa aaaaaagaag gaagaggagg agaagattca
     2641 atctcttaag gagaaaaatt cccagttgaa gcaagaaatt tcccagctgg aagagtaatc
     2701 tagagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca gcctcgactg
     2761 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2821 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2881 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2941 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3001 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3061 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3121 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3181 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3241 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccttttga
     3301 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3361 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3421 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3481 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3541 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3601 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3661 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3721 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     3781 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     3841 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3901 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3961 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4021 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4081 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4141 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4201 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4261 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4321 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4381 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4441 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4501 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4561 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4621 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4681 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4741 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4801 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4861 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4921 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4981 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5041 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5101 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5161 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5221 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5281 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5341 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5401 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5461 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5521 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5581 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5641 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5701 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5761 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5821 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5881 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5941 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6001 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6061 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6121 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6181 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6241 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6301 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6361 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6421 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6481 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6541 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6601 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6661 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6721 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6781 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6841 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6901 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6961 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7021 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7081 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7141 cgaaaagtgc cacctgacgt c
//
