LOCUS       sequence-199214-        6964 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 6964)
  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 6964)
  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: TIA1 RRM-CFP-FRBx5
FEATURES             Location/Qualifiers
     source          1..6964
                     /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      597..606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1266..1275
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1272..1274,1275..1277,1278..1988)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1317..1338)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1578..1597)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1925..1946
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2001..2279
                     /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             2370..2648
                     /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             2691..2969
                     /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             3012..3290
                     /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             3333..3611
                     /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    3752..3873
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3789..3808)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3843..3862
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3880..4335)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4017..4038)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4229..4248
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4362..4466
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4468..4825
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4468..4659
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4491..4511)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4676..4811
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4860..5654
                     /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             4860..5654
                     /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(4914..4933)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5524..5543
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5842..5861)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5886..5933
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6262..6850
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6751..6770
                     /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 ccatggagga cgagatgccc aagactctat acgtcggtaa cctttccaga gatgtgacag
      661 aagctctaat tctgcaactc tttagccaga ttggaccttg taaaaactgc aaaatgatta
      721 tggatacagc tggaaatgat ccctattgtt ttgtggagtt tcatgagcat cgtcatgcag
      781 ctgcagcatt agctgctatg aatggacgga agataatggg taaggaagtc aaagtgaatt
      841 gggcaacaac ccctagcagt caaaagaaag atacaagcaa tcatttccat gtctttgttg
      901 gtgatctcag cccagaaatt acaactgaag atataaaagc tgcttttgca ccatttggaa
      961 gaatatcaga tgcccgagtg gtaaaagaca tggcaacagg aaagtctaag ggatatggct
     1021 ttgtctcctt tttcaacaaa tgggatgctg aaaacgccat tcaacagatg ggtggccagt
     1081 ggcttggtgg aagacaaatc agaactaact gggcaacccg aaagcctccc gctccaaaga
     1141 gtacatatga gtcaaatacc aaacagctat catatgatga ggttgtaaat cagtctagtc
     1201 caagcaactg tactgtatac tgtggaggtg ttacttctgg gctaacagaa caactaggac
     1261 cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1321 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1381 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1441 cctggcccac cctcgtgacc accctgacct ggggcgtgca gtgcttcagc cgctaccccg
     1501 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1561 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1621 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1681 tcctggggca caagctggag tacaactaca tcagccacaa cgtctatatc accgccgaca
     1741 agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag gacggcagcg
     1801 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1861 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     1921 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1981 tgtacaaggg agcaagtgga atcctctggc atgagatgtg gcatgaaggc ctggaagagg
     2041 catctcgttt gtactttggg gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct
     2101 tgcatgctat gatggaacgg ggcccccaga ctctgaagga aacatccttt aatcaggcct
     2161 atggtcgaga tttaatggag gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg
     2221 tcaaggacct cctccaagcc tgggacctct attatcatgt gttccgacga atctcaaaga
     2281 gtgctggtgg tagtgctggt ggtagtgctg gtggtactgg tggtcctcga gctagtgctg
     2341 gtggtagtgc tggtggtagt gctggtggta tcctctggca tgagatgtgg catgaaggcc
     2401 tggaagaggc atctcgtttg tactttgggg aaaggaacgt gaaaggcatg tttgaggtgc
     2461 tggagccctt gcatgctatg atggaacggg gcccccagac tctgaaggaa acatccttta
     2521 atcaggccta tggtcgagat ttaatggagg cccaagagtg gtgcaggaag tacatgaaat
     2581 cagggaatgt caaggacctc ctccaagcct gggacctcta ttatcatgtg ttccgacgaa
     2641 tctcaaagaa gcttagtgct ggtggtagtg ctggtggtag tgctggtggt atcctctggc
     2701 atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg gaaaggaacg
     2761 tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg ggcccccaga
     2821 ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag gcccaagagt
     2881 ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cctccaagcc tgggacctct
     2941 attatcatgt gttccgacga atctcaaagg aattcagtgc tggtggtagt gctggtggta
     3001 gtgctggtgg tatcctctgg catgagatgt ggcatgaagg cctggaagag gcatctcgtt
     3061 tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta
     3121 tgatggaacg gggcccccag actctgaagg aaacatcctt taatcaggcc tatggtcgag
     3181 atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc
     3241 tcctccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag gtcgacagtg
     3301 ctggtggtag tgctggtggt agtgctggtg gtatcctctg gcatgagatg tggcatgaag
     3361 gcctggaaga ggcatctcgt ttgtactttg gggaaaggaa cgtgaaaggc atgtttgagg
     3421 tgctggagcc cttgcatgct atgatggaac ggggccccca gactctgaag gaaacatcct
     3481 ttaatcaggc ctatggtcga gatttaatgg aggcccaaga gtggtgcagg aagtacatga
     3541 aatcagggaa tgtcaaggac ctcctccaag cctgggacct ctattatcat gtgttccgac
     3601 gaatctcaaa gccgcgggcc cgggatccac cggatctaga taactgatca taatcagcca
     3661 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     3721 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     3781 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3841 ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt
     3901 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     3961 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     4021 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     4081 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     4141 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4201 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     4261 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     4321 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4381 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4441 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     4501 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4561 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4621 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4681 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4741 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     4801 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     4861 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4921 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4981 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     5041 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     5101 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     5161 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     5221 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     5281 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     5341 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     5401 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     5461 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     5521 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     5581 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     5641 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     5701 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     5761 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     5821 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     5881 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     5941 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     6001 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     6061 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     6121 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6181 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6241 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6301 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6361 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6421 gccgtagtta ggccaccact tcaagaactc tgtatcaccg cctacatacc tcgctctgct
     6481 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6541 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6601 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6661 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6721 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6781 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6841 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6901 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
     6961 gcat
//
