LOCUS       sequence-451382-        6774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (P6f)x3-(P14f)x3, with 
            short flexible linkers and N-term 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 6774)
  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 6774)
  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(P6f)3(P14f)3-gs
FEATURES             Location/Qualifiers
     source          1..6774
                     /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"
     CDS             1807..1818
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        complement(2327..2345)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2328..2345)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2365..2382)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2371..2595
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2641..3069
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2728..2747)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2938..2959
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3078..3098)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3083..3412
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3263..3398
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3325..3344
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3479..4273
                     /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(3533..3552)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4143..4162
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4447..4568
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4484..4503)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4538..4557
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4617..4633)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4617..4633)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4630..4652)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4641..4657
                     /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(4665..4671,4672..4689,4690..4695))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4710..4731
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4848..4865)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5019..5607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5099..5118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5778..6569,6570..6638))
                     /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     6401..6420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6639..6743)
                     /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 gctcaggctc
     1681 tggtgggagc aaaaacgcag ccttgaaaga agaaattgcg gctctggaag aggagaatgc
     1741 ggcccttgaa gaaaaaatag ctgcactgaa ggccggtagt ggaagtgggg gttcaaagaa
     1801 tgcggcattg aaggaagaga ttgctgcctt ggaggaggag aatgccgcgc tcgaggaaaa
     1861 gatcgcagct ttgaaagcgg gatctggatc aggtggcagc aaaaatgctg ctctgaaaga
     1921 ggaaatagca gctctggagg aggagaacgc cgccctcgag gaaaaaattg ccgccttgaa
     1981 agcgggcggc tctggcggcg gctccggagg ctctgaaatt gcagctctcg aggagaagat
     2041 tgcagccctg aagcaggaga atgcagccct ggaagagaaa aatgcggcgt tgaaggctgg
     2101 aagtggtagt gggggatccg aaattgctgc gttggaagag aagatagcgg cgcttaaaca
     2161 agaaaatgca gccctcgaag aaaaaaatgc tgcacttaaa gcggggtcag gatcaggtgg
     2221 ttcagagatc gccgctcttg aagagaaaat agccgcattg aagcaagaga acgcagcctt
     2281 ggaggaaaaa aacgctgcgc tgaaagcata atctagaggg ccctattcta tagtgtcacc
     2341 taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2401 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2461 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2521 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     2581 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     2641 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2701 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2761 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2821 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2881 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2941 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3001 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3061 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3121 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3181 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3241 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3301 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     3361 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     3421 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     3481 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3541 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3601 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3661 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3721 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3781 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3841 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3901 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3961 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     4021 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4081 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4141 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4201 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4261 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     4321 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4381 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4441 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4501 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4561 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4621 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4681 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4741 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4801 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4861 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4921 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4981 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5041 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5101 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5161 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5221 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5281 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5341 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5401 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5461 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5521 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5581 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5641 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5701 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5761 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5821 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5881 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5941 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6001 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6061 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6121 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6181 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6241 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6301 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6361 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6421 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6481 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6541 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6601 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6661 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6721 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
