LOCUS       sequence-454693-        6792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (P5f-P13f)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 6792)
  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 6792)
  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(P5f-P13f)3-gs
FEATURES             Location/Qualifiers
     source          1..6792
                     /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(2345..2363)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2346..2363)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2383..2400)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2389..2613
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2659..3087
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2746..2765)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2956..2977
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3096..3116)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3101..3430
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3281..3416
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3343..3362
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3497..4291
                     /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(3551..3570)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4161..4180
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4465..4586
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4502..4521)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4556..4575
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4635..4651)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4635..4651)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4648..4670)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4659..4675
                     /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(4683..4689,4690..4707,4708..4713))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4728..4749
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4866..4883)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5037..5625)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5117..5136)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5796..6587,6588..6656))
                     /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     6419..6438
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6657..6761)
                     /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 gtacaagggc tcaggctctg gtgggagcga gaacgccgca ctcgaggaga agatcgcagc
     1741 actgaaagag aaaaatgcag cgcttaaaga ggaaattgcg gccttggagg cgggctccgg
     1801 tagtgggggg agtaaaatag cagcgctcaa agaggagatc gctgcccttg aacagaaaaa
     1861 tgcagccctc aaagaagaaa acgcagcgct ggaagccggt tcagggtccg gcggaagcga
     1921 aaatgcggca ctggaagaaa agattgcggc actcaaagag aagaacgcgg ctctgaagga
     1981 agaaatcgcc gctcttgagg ctggcggctc tggcggcggc tccggaggct ctaagattgc
     2041 cgccctcaaa gaggagatcg ctgcattgga acaaaaaaat gcagctctga aggaagagaa
     2101 tgcggctctt gaagctggga gtgggtctgg gggcagcgag aacgctgctc tggaagaaaa
     2161 gatagccgct ttgaaagaaa aaaatgctgc tcttaaggag gaaatcgcag cactggaggc
     2221 tggctcaggc tcagggggga gcaaaatcgc cgcgttgaag gaagaaatag cggccctgga
     2281 gcaaaagaac gccgcgctga aagaagagaa tgcagccctt gaggcctaat ctagagggcc
     2341 ctattctata gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta
     2401 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2461 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2521 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2581 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     2641 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2701 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2761 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2821 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2881 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2941 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3001 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3061 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     3121 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3181 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3241 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3301 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3361 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3421 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     3481 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     3541 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     3601 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3661 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3721 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3781 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3841 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3901 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3961 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4021 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4081 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4141 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4201 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4261 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4321 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4381 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4441 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4501 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4561 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4621 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4681 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4741 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4801 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4861 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4921 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4981 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5041 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5101 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5161 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5221 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5281 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5341 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5401 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5461 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5521 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5581 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5641 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5701 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5761 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5821 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5881 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5941 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6001 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6061 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6121 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6181 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6241 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6301 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6361 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6421 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6481 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6541 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6601 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6661 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6721 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6781 ccacctgacg tc
//
