LOCUS       sequence-451399-        6531 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (D1h-D3h)x3, with short
            flexible linkers and N-terminal mCitrine; 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 6531)
  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 6531)
  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: mCit(D1h-D3h)3-gs
FEATURES             Location/Qualifiers
     source          1..6531
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..52)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     279..299
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(308..736)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(418..439)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     630..649
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    782..1006
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     995..1012
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        1032..1050
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1032..1049
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(1447..2157,2158..2160,2161..2163))
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNCIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1489..1510)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1838..1857
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2097..2118
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2164..2181)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2252..2271)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2253..2271)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2316..2519)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2345..2365)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2520..2899
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3071..3090
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3165..3269
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3270..3338,3339..4130)
                     /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(3488..3507)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4301..4889
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4790..4809
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5043..5060
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5177..5198
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5213..5218,5219..5236,5237..5243)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5251..5267
                     /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     5256..5278
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5275..5291
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5275..5291
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5340..5461
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5351..5370)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5405..5424
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5635..6429)
                     /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(5746..5765)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6356..6375
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6496..6531,1..294))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6510..6531,1..114)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ctacttctgg aatagctcag aggcagaggc ggcctcggcc tctgcataaa taaaaaaaat
       61 tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg
      121 agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc
      181 tgctggggag cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg
      241 catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca
      301 cagaattaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
      361 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
      421 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
      481 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
      541 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
      601 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
      661 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
      721 ccgctacagg gcgcgtgggg atacccccta gagccccagc tggttctttc cgcctcagaa
      781 gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc ctcccccttg
      841 ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca
      901 atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc
      961 acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag
     1021 cgagctctag catttaggtg acactataga atagggccct ctagattact cttccagcgc
     1081 agcattttct tgcttaagag cagctatttt agagccgcca gagcctgatc cttctttcag
     1141 ggctgcgttc ttctgttcaa gtgctgcaat ttcagaaccg ccagatccag agccctcctc
     1201 aagggcggca ttctcttgct tcaaagccgc gattttactg ccaccactac cagaaccctc
     1261 cttcagagcc gcgtttttct gctccagtgc agcaatttca ctgccgccag aaccactgcc
     1321 ttcttccaag gctgcgttct cctgtttcaa cgccgcaatt ttgctgcctc cagacccgct
     1381 gccctctttc agggccgcat tcttctgttc gagcgcagca atctcgctcc caccagagcc
     1441 tgagcccttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag
     1501 caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact ggtagctcag
     1561 gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg
     1621 gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat
     1681 gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt tgtactccag
     1741 cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcagtt
     1801 caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt
     1861 gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg
     1921 ctgcttcatg tggtcggggt agcgggcgaa gcacatcagg ccgtagccga aggtggtcac
     1981 gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagctt
     2041 gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc
     2101 gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac
     2161 catgtggtgg tggtggtggt gcatggtggc gaattccagc acactggcgg ccgttactag
     2221 tggatccgag ctcggtacca agcttgggtc tccctatagt gagtcgtatt aatttcgata
     2281 agccagtaag cagtgggttc tctagttagc cagagagctc tgcttatata gacctcccac
     2341 cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag
     2401 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
     2461 aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc
     2521 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
     2581 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
     2641 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
     2701 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
     2761 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
     2821 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
     2881 ctagtcaata atcaatgtca acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa
     2941 cgcctaaccc taagcagatt cttcatgcaa ttgtcggtca agccttgcct tgttgtagct
     3001 taaattttgc tcgcgcacta ctcagcgacc tccaacacac aagcagggag cagatactgg
     3061 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatagggg atcgggagat
     3121 ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3181 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3241 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3301 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3361 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3421 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3481 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3541 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3601 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3661 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3721 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3781 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3841 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3901 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3961 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     4021 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4081 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4141 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4201 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4261 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4321 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4381 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4441 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4501 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4561 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4621 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4681 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4741 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4801 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4861 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4921 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4981 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5041 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     5101 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     5161 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     5221 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     5281 acagctatga ccatgattac gccaagctct agctagaggt cgacggtata cagacatgat
     5341 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     5401 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     5461 tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt
     5521 cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc
     5581 gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa
     5641 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
     5701 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
     5761 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
     5821 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
     5881 atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc
     5941 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
     6001 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
     6061 cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga
     6121 caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac
     6181 aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc
     6241 ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg
     6301 cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca
     6361 gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg
     6421 ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatccgaaaa tggatataca
     6481 agctcccggg agctttttgc aaaagcctag gcctccaaaa aagcctcctc a
//
