LOCUS       sequence-196604-        6367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses iPOLYMER component in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6367)
  AUTHORS   Nakamura H, Lee AA, Afshar AS, Watanabe S, Rho E, Razavi S, Suarez 
            A, Lin YC, Tanigawa M, Huang B, DeRose R, Bobb D, Hong W, Gabelli 
            SB, Goutsias J, Inoue T
  TITLE     Intracellular production of hydrogels and synthetic RNA granules by 
            multivalent molecular interactions.
  JOURNAL   Nat Mater. 2017 Nov 6. pii: nmat5006. doi: 10.1038/nmat5006.
  PUBMED    29115293
REFERENCE   2  (bases 1 to 6367)
  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 Mater. 
            2017 Nov 6. pii: nmat5006. doi: 10.1038/nmat5006."
COMMENT     Sequence Label: cyto-CFP-FRBx5
FEATURES             Location/Qualifiers
     source          1..6367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      669..678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(675..677,678..680,681..1391)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(720..741)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(981..1000)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1328..1349
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1404..1682
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             1773..2051
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             2094..2372
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             2415..2693
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             2736..3014
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     polyA_signal    3155..3276
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3192..3211)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3246..3265
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3283..3738)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3420..3441)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3632..3651
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3765..3869
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3871..4228
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3871..4062
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3894..3914)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4079..4214
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4263..5057
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4263..5057
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4317..4336)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4927..4946
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5245..5264)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5289..5336
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5665..6253
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6154..6173
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcca
      601 ccatgaacct ggtggacctc caaaagaagc tggaggagct ggagctggac gagcagcagg
      661 gaccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      721 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      781 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      841 tgccctggcc caccctcgtg accaccctga cctggggcgt gcagtgcttc agccgctacc
      901 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      961 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1021 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1081 acatcctggg gcacaagctg gagtacaact acatcagcca caacgtctat atcaccgccg
     1141 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggca
     1201 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1261 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     1321 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1381 agctgtacaa gggagcaagt ggaatcctct ggcatgagat gtggcatgaa ggcctggaag
     1441 aggcatctcg tttgtacttt ggggaaagga acgtgaaagg catgtttgag gtgctggagc
     1501 ccttgcatgc tatgatggaa cggggccccc agactctgaa ggaaacatcc tttaatcagg
     1561 cctatggtcg agatttaatg gaggcccaag agtggtgcag gaagtacatg aaatcaggga
     1621 atgtcaagga cctcctccaa gcctgggacc tctattatca tgtgttccga cgaatctcaa
     1681 agagtgctgg tggtagtgct ggtggtagtg ctggtggtac tggtggtcct cgagctagtg
     1741 ctggtggtag tgctggtggt agtgctggtg gtatcctctg gcatgagatg tggcatgaag
     1801 gcctggaaga ggcatctcgt ttgtactttg gggaaaggaa cgtgaaaggc atgtttgagg
     1861 tgctggagcc cttgcatgct atgatggaac ggggccccca gactctgaag gaaacatcct
     1921 ttaatcaggc ctatggtcga gatttaatgg aggcccaaga gtggtgcagg aagtacatga
     1981 aatcagggaa tgtcaaggac ctcctccaag cctgggacct ctattatcat gtgttccgac
     2041 gaatctcaaa gaagcttagt gctggtggta gtgctggtgg tagtgctggt ggtatcctct
     2101 ggcatgagat gtggcatgaa ggcctggaag aggcatctcg tttgtacttt ggggaaagga
     2161 acgtgaaagg catgtttgag gtgctggagc ccttgcatgc tatgatggaa cggggccccc
     2221 agactctgaa ggaaacatcc tttaatcagg cctatggtcg agatttaatg gaggcccaag
     2281 agtggtgcag gaagtacatg aaatcaggga atgtcaagga cctcctccaa gcctgggacc
     2341 tctattatca tgtgttccga cgaatctcaa aggaattcag tgctggtggt agtgctggtg
     2401 gtagtgctgg tggtatcctc tggcatgaga tgtggcatga aggcctggaa gaggcatctc
     2461 gtttgtactt tggggaaagg aacgtgaaag gcatgtttga ggtgctggag cccttgcatg
     2521 ctatgatgga acggggcccc cagactctga aggaaacatc ctttaatcag gcctatggtc
     2581 gagatttaat ggaggcccaa gagtggtgca ggaagtacat gaaatcaggg aatgtcaagg
     2641 acctcctcca agcctgggac ctctattatc atgtgttccg acgaatctca aaggtcgaca
     2701 gtgctggtgg tagtgctggt ggtagtgctg gtggtatcct ctggcatgag atgtggcatg
     2761 aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg
     2821 aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg aaggaaacat
     2881 cctttaatca ggcctatggt cgagatttaa tggaggccca agagtggtgc aggaagtaca
     2941 tgaaatcagg gaatgtcaag gacctcctcc aagcctggga cctctattat catgtgttcc
     3001 gacgaatctc aaagccgcgg gcccgggatc caccggatct agataactga tcataatcag
     3061 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     3121 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     3181 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3241 tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata
     3301 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     3361 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     3421 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     3481 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     3541 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     3601 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     3661 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     3721 cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3781 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3841 tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt
     3901 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3961 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     4021 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     4081 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4141 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4201 cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc
     4261 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     4321 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     4381 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     4441 tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     4501 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     4561 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     4621 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     4681 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     4741 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg
     4801 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     4861 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     4921 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     4981 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     5041 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     5101 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     5161 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5221 cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     5281 cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa
     5341 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
     5401 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
     5461 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat
     5521 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5581 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5641 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5701 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5761 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     5821 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5881 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5941 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6001 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6061 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6121 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6181 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6241 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6301 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6361 catgcat
//
