LOCUS       sequence-196623-        8018 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 8018)
  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 8018)
  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: pMT2 TIA1 RRM-CFP-FRBx5
FEATURES             Location/Qualifiers
     source          1..8018
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(7..336)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      21..156
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     321..341
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     intron          805..1034
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1017..1036
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1062..1071
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1731..1740
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1737..1739,1740..1742,1743..2453)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1782..1803)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2043..2062)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2390..2411
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2466..2744
                     /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             2835..3113
                     /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             3156..3434
                     /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             3477..3755
                     /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             3798..4076
                     /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    4969..5103
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5006..5025)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5060..5079
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5630..5647)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5801..6389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5881..5900)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6560..7351,7352..7420))
                     /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     7183..7202
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7421..7525)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7593..7611
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7649..7671)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7771..7790
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7984..8006
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7998..8015
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7999..8015
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc
       61 agaggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg
      121 agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat
      181 ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga
      241 ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
      301 ggagcctggg gactttccac accctaactg acacacattc cacaggatcc ggtcgcgcga
      361 atttcgagcg gtgttccgcg gtcctcctcg tatagaaact cggaccactc tgagacgaag
      421 gctcgcgtcc aggccagcac gaaggaggct aagtgggagg ggtagcggtc gttgtccact
      481 agggggtcca ctcgctccag ggtgtgaaga cacatgtcgc cctcttcggc atcaaggaag
      541 gtgattggtt tataggtgta ggccacgtga ccgggtgttc ctgaaggggg gctataaaag
      601 ggggtggggg cgcgttcgtc ctcactctct tccgcatcgc tgtctgcgag ggccagctgt
      661 tgggctcgcg gttgaggaca aactcttcgc ggtctttcca gtactcttgg atcggaaacc
      721 cgtcggcctc cgaacggtac tccgccaccg agggacctga gcgagtccgc atcgaccgga
      781 tcggaaaacc tctcgactgt tggggtgagt actccctctc aaaagcgggc atgacttctg
      841 cgctaagatt gtcagtttcc aaaaacgagg aggatttgat attcacctgg cccgcggtga
      901 tgcctttgag ggtggccgcg tccatctggt cagaaaagac aatctttttg ttgtcaagct
      961 tgaggtgtgg caggcttgag atctggccat acacttgagt gacaatgaca tccactttgc
     1021 ctttctctcc acaggtgtcc actcccaggt ccaactgcag ggccaccatg gaggacgaga
     1081 tgcccaagac tctatacgtc ggtaaccttt ccagagatgt gacagaagct ctaattctgc
     1141 aactctttag ccagattgga ccttgtaaaa actgcaaaat gattatggat acagctggaa
     1201 atgatcccta ttgttttgtg gagtttcatg agcatcgtca tgcagctgca gcattagctg
     1261 ctatgaatgg acggaagata atgggtaagg aagtcaaagt gaattgggca acaaccccta
     1321 gcagtcaaaa gaaagataca agcaatcatt tccatgtctt tgttggtgat ctcagcccag
     1381 aaattacaac tgaagatata aaagctgctt ttgcaccatt tggaagaata tcagatgccc
     1441 gagtggtaaa agacatggca acaggaaagt ctaagggata tggctttgtc tcctttttca
     1501 acaaatggga tgctgaaaac gccattcaac agatgggtgg ccagtggctt ggtggaagac
     1561 aaatcagaac taactgggca acccgaaagc ctcccgctcc aaagagtaca tatgagtcaa
     1621 ataccaaaca gctatcatat gatgaggttg taaatcagtc tagtccaagc aactgtactg
     1681 tatactgtgg aggtgttact tctgggctaa cagaacaact aggaccggtc gccaccatgg
     1741 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1801 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1861 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1921 tgaccaccct gacctggggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     1981 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2041 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2101 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2161 tggagtacaa ctacatcagc cacaacgtct atatcaccgc cgacaagcag aagaacggca
     2221 tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2281 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2341 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2401 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggagcaa
     2461 gtggaatcct ctggcatgag atgtggcatg aaggcctgga agaggcatct cgtttgtact
     2521 ttggggaaag gaacgtgaaa ggcatgtttg aggtgctgga gcccttgcat gctatgatgg
     2581 aacggggccc ccagactctg aaggaaacat cctttaatca ggcctatggt cgagatttaa
     2641 tggaggccca agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag gacctcctcc
     2701 aagcctggga cctctattat catgtgttcc gacgaatctc aaagagtgct ggtggtagtg
     2761 ctggtggtag tgctggtggt actggtggtc ctcgagctag tgctggtggt agtgctggtg
     2821 gtagtgctgg tggtatcctc tggcatgaga tgtggcatga aggcctggaa gaggcatctc
     2881 gtttgtactt tggggaaagg aacgtgaaag gcatgtttga ggtgctggag cccttgcatg
     2941 ctatgatgga acggggcccc cagactctga aggaaacatc ctttaatcag gcctatggtc
     3001 gagatttaat ggaggcccaa gagtggtgca ggaagtacat gaaatcaggg aatgtcaagg
     3061 acctcctcca agcctgggac ctctattatc atgtgttccg acgaatctca aagaagctta
     3121 gtgctggtgg tagtgctggt ggtagtgctg gtggtatcct ctggcatgag atgtggcatg
     3181 aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg
     3241 aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg aaggaaacat
     3301 cctttaatca ggcctatggt cgagatttaa tggaggccca agagtggtgc aggaagtaca
     3361 tgaaatcagg gaatgtcaag gacctcctcc aagcctggga cctctattat catgtgttcc
     3421 gacgaatctc aaaggaattc agtgctggtg gtagtgctgg tggtagtgct ggtggtatcc
     3481 tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa
     3541 ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg gaacggggcc
     3601 cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta atggaggccc
     3661 aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcctc caagcctggg
     3721 acctctatta tcatgtgttc cgacgaatct caaaggtcga cagtgctggt ggtagtgctg
     3781 gtggtagtgc tggtggtatc ctctggcatg agatgtggca tgaaggcctg gaagaggcat
     3841 ctcgtttgta ctttggggaa aggaacgtga aaggcatgtt tgaggtgctg gagcccttgc
     3901 atgctatgat ggaacggggc ccccagactc tgaaggaaac atcctttaat caggcctatg
     3961 gtcgagattt aatggaggcc caagagtggt gcaggaagta catgaaatca gggaatgtca
     4021 aggacctcct ccaagcctgg gacctctatt atcatgtgtt ccgacgaatc tcaaagccgc
     4081 gggcccggga tccaccggat ctagataact gacaattcgg gggggggggg gggggggaca
     4141 gctcagggct gcgatttcgc gccaaacttg acggcaatcc tagcgtgaag gctggtagga
     4201 ttttatcccc gctgccatca tggttcgacc attgaactgc atcgtcgccg tgtcccaaaa
     4261 tatggggatt ggcaagaacg gagacctacc ctggcctccg ctcaggaacg agttcaagta
     4321 cttccaaaga atgaccacaa cctcttcagt ggaaggtaaa cagaatctgg tgattatggg
     4381 taggaaaacc tggttctcca ttcctgagaa gaatcgacct ttaaaggaca gaattaatat
     4441 agttctcagt agagaactca aagaaccacc acgaggagct cattttcttg ccaaaagttt
     4501 ggatgatgcc ttaagactta ttgaacaacc ggaattggca agtaaagtag acatggttgg
     4561 atagtcggag gcagttctgt ttaccaggaa gccatgaatc aaccaggcca cctcagactc
     4621 tttgtgacaa ggatcatgca ggaatttgaa agtgacacgt ttttcccaga aattgatttg
     4681 gggaaatata aacttctccc agaataccca ggcgtcctct ctgaggtcca ggaggaaaaa
     4741 ggcatcaagt ataagtttga agtctacgag aagaaagact aacaggaaga tgctttcaag
     4801 ttctctgctc ccctcctaaa gctatgcatt tttataagac catgggactt ttgctggctt
     4861 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     4921 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     4981 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5041 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     5101 atccccggcc aacggtctgg tgacccggct gcgagagctc ggtgtacctg agacgcgagt
     5161 aagcccttga gtcaaagacg tagtcgttgc aagtccgcac caggtactga tatcccacca
     5221 aaaagtgcgg cggcggctgg cggtagaggg gccagcgtag ggtggccggg gctccggggg
     5281 cgaggtcttc caacataagg cgatgatcat cgatgctaga ccgtgcaaaa ggagagcctg
     5341 taagcgggca ctcttccgtg gtctggtgga taaattcgca agggtatcat ggcggacgac
     5401 cggggttcga accccggatc cggccgtccg ccgtgatcca tgcggttacc gcccgcgtgt
     5461 cgaacccagg tgtgcgacgt cagacaacgg gggagcgctc cttttggctt ccttccaggc
     5521 gcggcggctg ctgcgctagc ttttttggcg agctcgaatt aattctgcat taatgaatcg
     5581 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5641 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5701 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5761 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5821 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5881 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5941 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6001 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6061 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6121 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6181 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6241 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6301 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6361 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6421 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6481 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6541 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6601 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6661 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6721 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6781 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6841 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6901 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6961 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7021 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7081 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7141 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7201 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7261 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7321 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7381 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7441 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7501 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     7561 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     7621 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     7681 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     7741 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     7801 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
     7861 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     7921 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     7981 tttcccagtc acgacgttgt aaaacgacgg ccagtgcc
//
