LOCUS       sequence-451397-        7179 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (S1-S3)x3, with long 
            rigid 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 7179)
  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 7179)
  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(S1-S3)3-SAH
FEATURES             Location/Qualifiers
     source          1..7179
                     /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             join(971..973,974..976,977..1687)
                     /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(1016..1037)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1277..1296)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1624..1645
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2732..2750)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2733..2750)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2770..2787)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2776..3000
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3046..3474
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3133..3152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3343..3364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3483..3503)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3488..3817
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3668..3803
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3730..3749
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3884..4678
                     /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(3938..3957)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4548..4567
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4852..4973
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4889..4908)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4943..4962
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5022..5038)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5022..5038)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5035..5057)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5046..5062
                     /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(5070..5076,5077..5094,5095..5100))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5115..5136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5253..5270)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5424..6012)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5504..5523)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6183..6974,6975..7043))
                     /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     6806..6825
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7044..7148)
                     /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 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1021 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1081 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1141 ctggcccacc ctcgtgacca ccttcggcta cggcctgatg tgcttcgccc gctaccccga
     1201 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1261 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1321 cgacaccctg gtgaactgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1381 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1441 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1501 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1561 cgacaaccac tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga
     1621 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1681 gtacaaggaa gaagaggaga agaaaaagaa agaggaggag gagaagaaga aaaaagaaga
     1741 agaggaaaaa aaaaagaaag aggaggagga gaagaaaaag aaagaagagg aagagaaaat
     1801 ccagtccctg aaagaggaaa ttagtcaatt ggagcagaaa aattctcagc tcaaagaaga
     1861 ggaggaggaa aagaaaaaga aggaggaaga ggaaaagaag aaaaaggagg aagaagagaa
     1921 gaagaagaag gaagaggaag agaagaaaaa gaaagaagaa gaggaagaga ttcaaagtct
     1981 ggaagaggag aacagtcaac tggagcagaa gatatctcag ctcaaggagg aagaggagga
     2041 aaagaaaaaa aaggaagaag aagaaaaaaa gaaaaaggaa gaggaggaga aaaaaaaaaa
     2101 agaagaagaa gagaagaaga aaaaggagga agaggagaag atccaaagtc tgaaggaaga
     2161 gatatcccaa ctcgaacaaa aaaatagtca gctgaaagag gaagaagagg agaaaaaaaa
     2221 gaaggaagag gaagaaaaga aaaaaaagga agaagaagaa aaaaaaaaaa aggaagaaga
     2281 agaaaaaaag aagaaagagg aggaagagga aatacaatcc ttggaagagg aaaattctca
     2341 gctggaacag aagatctcac agctcaaaga ggaggaggaa gagaagaaga agaaggagga
     2401 ggaggaaaaa aaaaaaaagg aggaggaaga aaaaaagaag aaagaagagg aagaaaagaa
     2461 gaagaaagaa gaagaggaga aaatccagtc acttaaagaa gagatttccc aactcgaaca
     2521 aaagaattcc cagctcaagg aggaggaaga ggaaaagaaa aagaaagaag aggaagaaaa
     2581 gaagaagaag gaggaagagg agaaaaagaa gaaggaggag gaggaaaaaa agaagaaaga
     2641 ggaggaagag gaaatccagt cattggagga agagaactca cagttggaac aaaagataag
     2701 ccaactcaag gagtaatcta gagggcccta ttctatagtg tcacctaaat gctagagctc
     2761 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2821 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2881 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2941 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     3001 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     3061 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3121 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3181 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3241 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3301 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3361 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3421 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     3481 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3541 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3601 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3661 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3721 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     3781 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     3841 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     3901 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     3961 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     4021 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     4081 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     4141 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     4201 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     4261 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     4321 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     4381 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     4441 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     4501 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     4561 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     4621 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     4681 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     4741 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4801 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4861 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4921 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4981 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5041 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5101 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5161 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5221 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5281 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5341 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5401 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5461 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5521 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5581 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5641 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5701 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5761 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5821 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5881 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5941 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6001 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6061 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6121 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6181 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6241 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6301 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6361 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6421 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6481 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6541 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6601 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6661 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6721 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6781 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6841 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6901 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6961 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7021 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7081 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7141 gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
