LOCUS       sequence-451170-        6822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (S1h)x8, 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 6822)
  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 6822)
  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(S1h)8-gs
FEATURES             Location/Qualifiers
     source          1..6822
                     /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(2375..2393)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2376..2393)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2413..2430)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2419..2643
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2689..3117
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2776..2795)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2986..3007
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3126..3146)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3131..3460
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3311..3446
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3373..3392
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3527..4321
                     /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(3581..3600)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4191..4210
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4495..4616
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4532..4551)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4586..4605
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4665..4681)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4665..4681)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4678..4700)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4689..4705
                     /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(4713..4719,4720..4737,4738..4743))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4758..4779
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4896..4913)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5067..5655)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5147..5166)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5826..6617,6618..6686))
                     /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     6449..6468
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6687..6791)
                     /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 gtgggagcaa aattgcggcg cttaaggaag agattgcagc
     1741 tttggagcaa aaaaacgctg cgttgaaaga gggatcaggg tctggtggaa gtaaaattgc
     1801 ggcacttaag gaagaaattg cagccttgga gcagaagaac gcagccctca aggaaggcag
     1861 cggttctggt gggtctaaaa tcgctgcgct taaagaagag atagcggcat tggaacagaa
     1921 aaatgcggcc cttaaggagg gcagtggatc tggcggctct aagatagccg ccttgaagga
     1981 agagattgcc gcactggagc aaaaaaatgc agcactcaaa gagggcggct ctggaggctc
     2041 taaaattgcg gcgcttaagg aagagattgc agctttggag caaaaaaacg ctgcgttgaa
     2101 agagggatca gggtctggtg gaagtaaaat tgcggcactt aaggaagaaa ttgcagcctt
     2161 ggagcagaag aacgcagccc tcaaggaagg cagcggttct ggtgggtcta aaatcgctgc
     2221 gcttaaagaa gagatagcgg cattggaaca gaaaaatgcg gcccttaagg agggcagtgg
     2281 atctggcggc tctaagatag ccgccttgaa ggaagagatt gccgcactgg agcaaaaaaa
     2341 tgcagcactc aaagagtaat ctagagggcc ctattctata gtgtcaccta aatgctagag
     2401 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2461 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2521 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2581 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2641 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2701 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2761 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2821 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2881 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2941 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3001 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3061 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3121 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3181 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3241 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3301 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3361 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3421 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3481 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3541 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3601 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3661 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3721 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3781 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3841 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3901 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3961 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4021 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     4081 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4141 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4201 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4261 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4321 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4381 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4441 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4501 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4561 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4621 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4681 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4741 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4801 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4861 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4921 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4981 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5041 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5101 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5161 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5221 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5281 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5341 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5401 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5461 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5521 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5581 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5641 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5701 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5761 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5821 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5881 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5941 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6001 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6061 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6121 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6181 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6241 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6301 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6361 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6421 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6481 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6541 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6601 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6661 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6721 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6781 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
