LOCUS       sequence-451384-        6495 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of coiled coil segments (S1-S3)x1, 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 6495)
  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 6495)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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)1-SAH
FEATURES             Location/Qualifiers
     source          1..6495
                     /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(2048..2066)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2049..2066)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2086..2103)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2092..2316
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2362..2790
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2449..2468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2659..2680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2799..2819)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2804..3133
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2984..3119
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3046..3065
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3200..3994
                     /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(3254..3273)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3864..3883
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4168..4289
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4205..4224)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4259..4278
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4338..4354)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4338..4354)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4351..4373)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4362..4378
                     /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(4386..4392,4393..4410,4411..4416))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4431..4452
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4569..4586)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4740..5328)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4820..4839)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5499..6290,6291..6359))
                     /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     6122..6141
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6360..6464)
                     /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 ctcaaggagt aatctagagg
     2041 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     2101 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2161 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2221 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2281 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     2341 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2401 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2461 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2521 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2581 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2641 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2701 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2761 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     2821 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2881 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2941 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3001 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3061 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3121 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     3181 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     3241 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     3301 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     3361 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     3421 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     3481 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     3541 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     3601 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     3661 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     3721 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     3781 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     3841 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     3901 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     3961 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4021 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4081 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4141 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4201 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4261 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4321 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4381 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4441 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4501 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4561 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4621 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4681 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4741 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4801 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4861 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4921 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4981 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5041 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5101 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5161 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5221 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5281 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5341 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5401 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5461 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5521 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5581 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5641 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5701 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5761 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5821 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5881 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5941 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6001 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6061 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6121 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6181 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6241 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6301 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6361 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6421 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6481 gtgccacctg acgtc
//
