LOCUS       sequence-454673-        6057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (P6f)x3-(P14f)x3, with 
            short flexible linkers; 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 6057)
  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 6057)
  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: (P6f)3(P14f)3-gs
FEATURES             Location/Qualifiers
     source          1..6057
                     /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"
     regulatory      944..953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1090..1101
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        complement(1610..1628)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1611..1628)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1648..1665)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1654..1878
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1924..2352
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2011..2030)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2221..2242
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2361..2381)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2366..2695
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2546..2681
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2608..2627
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2762..3556
                     /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(2816..2835)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3426..3445
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3730..3851
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3767..3786)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3821..3840
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3900..3916)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3900..3916)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3913..3935)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3924..3940
                     /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(3948..3954,3955..3972,3973..3978))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3993..4014
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4131..4148)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4302..4890)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4382..4401)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5061..5852,5853..5921))
                     /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     5684..5703
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5922..6026)
                     /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 ctccaccaca 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 tgggctcagg
      961 ctctggtggg agcaaaaacg cagccttgaa agaagaaatt gcggctctgg aagaggagaa
     1021 tgcggccctt gaagaaaaaa tagctgcact gaaggccggt agtggaagtg ggggttcaaa
     1081 gaatgcggca ttgaaggaag agattgctgc cttggaggag gagaatgccg cgctcgagga
     1141 aaagatcgca gctttgaaag cgggatctgg atcaggtggc agcaaaaatg ctgctctgaa
     1201 agaggaaata gcagctctgg aggaggagaa cgccgccctc gaggaaaaaa ttgccgcctt
     1261 gaaagcgggc ggctctggcg gcggctccgg aggctctgaa attgcagctc tcgaggagaa
     1321 gattgcagcc ctgaagcagg agaatgcagc cctggaagag aaaaatgcgg cgttgaaggc
     1381 tggaagtggt agtgggggat ccgaaattgc tgcgttggaa gagaagatag cggcgcttaa
     1441 acaagaaaat gcagccctcg aagaaaaaaa tgctgcactt aaagcggggt caggatcagg
     1501 tggttcagag atcgccgctc ttgaagagaa aatagccgca ttgaagcaag agaacgcagc
     1561 cttggaggaa aaaaacgctg cgctgaaagc ataatctaga gggccctatt ctatagtgtc
     1621 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     1681 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     1741 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     1801 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     1861 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     1921 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1981 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2041 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2101 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2161 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2221 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2281 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2341 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2401 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2461 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2521 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     2581 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     2641 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     2701 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     2761 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     2821 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     2881 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     2941 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3001 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3061 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3121 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3181 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3241 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     3301 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3361 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3421 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3481 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     3541 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     3601 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     3661 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     3721 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3781 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3841 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     3901 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     3961 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4021 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4081 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4141 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4201 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4261 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4321 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4381 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4441 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4501 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4561 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4621 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4681 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4741 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4801 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4861 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4921 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4981 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5041 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5101 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5161 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5221 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5281 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5341 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5401 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5461 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5521 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5581 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5641 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5701 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5761 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5821 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5881 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5941 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6001 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
