LOCUS       sequence-451401-        6483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (S2)x2-(S4)x2, 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 6483)
  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 6483)
  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)2(S4)2-gs
FEATURES             Location/Qualifiers
     source          1..6483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(63..82)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     673..692
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(813..1142)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      827..962
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(881..900)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1127..1147
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1156..1584)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1266..1287)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1478..1497
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1630..1854
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1843..1860
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        1880..1898
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1880..1897
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(2265..2963)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             complement(2964..2981)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3052..3071)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3053..3071)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3116..3319)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3145..3165)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3320..3699
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3871..3890
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3965..4069
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4070..4138,4139..4930)
                     /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     complement(4288..4307)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5101..5689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5590..5609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5843..5860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5977..5998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6013..6018,6019..6036,6037..6043)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6051..6067
                     /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)."
     primer_bind     6056..6078
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6075..6091
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6075..6091
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    6140..6261
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6151..6170)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6205..6224
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(6435..6483,1..746))
                     /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"
ORIGIN
        1 ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca
       61 gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca
      121 cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg
      181 ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt
      241 tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct
      301 tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta
      361 gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca
      421 ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc
      481 cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc
      541 cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg
      601 acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg
      661 attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct
      721 gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat
      781 ccgaaaatgg atatacaagc tcccgggagc tttttgcaaa agcctaggcc tccaaaaaag
      841 cctcctcact acttctggaa tagctcagag gcagaggcgg cctcggcctc tgcataaata
      901 aaaaaaatta gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg
      961 atgggcggag ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata
     1021 cttctgcctg ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc
     1081 atgctttgca tacttctgcc tgctggggag cctggggact ttccacaccc taactgacac
     1141 acattccaca gaattaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     1201 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     1261 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     1321 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     1381 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     1441 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     1501 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     1561 ttaatgcgcc gctacagggc gcgtggggat accccctaga gccccagctg gttctttccg
     1621 cctcagaagc catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct
     1681 cccccttgct gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat
     1741 gcgatgcaat ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca
     1801 aggaaggcac gggggagggg caaacaacag atggctggca actagaaggc acagtcgagg
     1861 ctgatcagcg agctctagca tttaggtgac actatagaat agggccctct agattactct
     1921 tccaactgag aaatctcttg tttgagctgg gagttcttct ctttaagact ttgtattttc
     1981 ccgctcccgg ggcttccgga accctcttca agctggctaa tttcctgttt cagttgtgag
     2041 ttcttttctt tcaaactttg tatcttgccg ctgcccgggc taccactgcc ctcctcaagt
     2101 tggctgtttt cctgcttcaa ttgacttatt ttctcttcga gagattgaat ttctccactt
     2161 cccggactcc cggaaccttc ctccaattga gagttctcct gcttaagctg acttatcttc
     2221 tcctcaagac tttgtatctc accgctccct ggggagccac tgccattgag cttgtgcccc
     2281 agtttgctag ggaggtcgca gtatctggcc actgccacct cgtgctgctc gacgtaggtc
     2341 tcgttgttgg cctccttgat tctttccagt ctgtagtcca catagtagac gccaggcatc
     2401 ttgaggttct tagcgggttt cttggatcta tatgtggtct tgatgtttgc gatcagatgg
     2461 ctcccgccca cgagcttcag ggccatgtcg tttctgcctt ccaggccgcc gtcagcgggg
     2521 tacagcgtct cggtgaaggc ctcccagccg agtgttttct tctgcatcac agggccgttg
     2581 gatgtgaagt tcacccctct gatcttgacg ttgtagatga ggcagccgtc ctggaggctg
     2641 gtgtcctggg tagcggtcag cacgcccccg tcttcgtatg tggtgactct ctcccatgtg
     2701 aagccctcag ggaaggactg cttgaagaag tcggggatgc cctgggtgtg gttgatgaag
     2761 gtcttgctgc cgtagaggaa gctagtagcc aggatgtcga aggcgaaggg gagagggccg
     2821 ccctcgacca ccttgattct catggtctgg gtgccctcgt agggcttgcc ttcgccctcg
     2881 gatgtgcact tgaagtgatg gttgtccacg gtgccctcca tgtacagctt catgtgcatg
     2941 ttctccttaa tcagctcgct catgtggtgg tggtggtggt gcatggtggc gaattccagc
     3001 acactggcgg ccgttactag tggatccgag ctcggtacca agcttgggtc tccctatagt
     3061 gagtcgtatt aatttcgata agccagtaag cagtgggttc tctagttagc cagagagctc
     3121 tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt
     3181 tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt
     3241 caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt
     3301 actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag
     3361 taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
     3421 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
     3481 tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg
     3541 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
     3601 catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg
     3661 taattgatta ctattaataa ctagtcaata atcaatgtca acgcgtatat ctggcccgta
     3721 catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa ttgtcggtca
     3781 agccttgcct tgttgtagct taaattttgc tcgcgcacta ctcagcgacc tccaacacac
     3841 aagcagggag cagatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     3901 accatagggg atcgggagat ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa
     3961 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4021 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4081 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4141 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4201 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4261 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     4321 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     4381 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     4441 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4501 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4561 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     4621 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     4681 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     4741 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     4801 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     4861 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     4921 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     4981 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5041 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5101 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5161 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5221 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5281 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5341 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5401 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5461 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5521 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5581 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5641 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5701 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5761 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     5821 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
     5881 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     5941 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
     6001 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
     6061 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     6121 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     6181 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     6241 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     6301 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     6361 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     6421 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     6481 tcg
//
