LOCUS       sequence-454692-        6825 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of coiled coil segments (S2h)x8, 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 6825)
  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 6825)
  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(S2h)8-gs
FEATURES             Location/Qualifiers
     source          1..6825
                     /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             971..1669
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     promoter        complement(2378..2396)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2379..2396)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2416..2433)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2422..2646
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2692..3120
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2779..2798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2989..3010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3129..3149)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3134..3463
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3314..3449
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3376..3395
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3530..4324
                     /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(3584..3603)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4194..4213
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4498..4619
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4535..4554)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4589..4608
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4668..4684)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4668..4684)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4681..4703)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4692..4708
                     /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(4716..4722,4723..4740,4741..4746))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4761..4782
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4899..4916)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5070..5658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5150..5169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5829..6620,6621..6689))
                     /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     6452..6471
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6690..6794)
                     /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 atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     1021 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     1081 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     1141 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
     1201 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     1261 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     1321 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     1381 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
     1441 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     1501 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     1561 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     1621 agtggccaga tactgcgacc tccctagcaa actggggcac aagctcaatg gctcaggctc
     1681 tggtgggagc gagatagctg cacttgagga gaaaatcgcg gcgctgaaac aggagaatgc
     1741 ggcactcgaa gaagggagtg gatctggtgg ctccgagatc gccgccttgg aagagaagat
     1801 tgcagccctc aaacaagaga acgctgctct ggaggaagga tctggctccg gtggatcaga
     1861 gatagccgcg cttgaagaga aaatagcagc acttaagcaa gagaacgcag cgctggagga
     1921 gggaagtgga agtggtggga gcgaaatagc ggcgttggag gagaagattg ccgcactgaa
     1981 acaagagaat gcagctctgg aagagggcgg ctctggaggc tctggctcag gctctggtgg
     2041 gagcgagata gctgcacttg aggagaaaat cgcggcgctg aaacaggaga atgcggcact
     2101 cgaagaaggg agtggatctg gtggctccga gatcgccgcc ttggaagaga agattgcagc
     2161 cctcaaacaa gagaacgctg ctctggagga aggatctggc tccggtggat cagagatagc
     2221 cgcgcttgaa gagaaaatag cagcacttaa gcaagagaac gcagcgctgg aggagggaag
     2281 tggaagtggt gggagcgaaa tagcggcgtt ggaggagaag attgccgcac tgaaacaaga
     2341 gaatgcagct ctggaagagt aatctagagg gccctattct atagtgtcac ctaaatgcta
     2401 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2461 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2521 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2581 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     2641 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     2701 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2761 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2821 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2881 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     2941 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3001 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3061 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3121 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3181 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3241 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3301 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3361 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     3421 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     3481 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3541 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3601 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     3661 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     3721 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     3781 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     3841 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     3901 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     3961 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4021 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4081 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4141 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4201 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4261 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4321 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4381 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4441 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     4501 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4561 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4621 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     4681 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4741 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4801 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4861 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4921 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4981 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5041 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5101 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5161 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5221 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5281 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5341 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5401 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5461 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5521 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5581 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5641 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5701 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5761 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5821 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5881 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5941 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6001 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6061 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6121 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6181 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6241 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6301 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6361 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6421 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6481 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6541 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6601 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6661 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6721 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6781 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
