LOCUS       sequence-351705-        6497 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a protein of interest fused to the 
            C-terminus of PB1-AzamiGreen-FRB-TEVuncs-mTagBFP-HA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6497)
  AUTHORS   Yoshikawa M, Yoshii T, Ikuta M, Tsukiji S
  TITLE     Synthetic Protein Condensates That Inducibly Recruit and Release 
            Protein Activity in Living Cells.
  JOURNAL   J Am Chem Soc. 2021 May 5;143(17):6434-6446. doi: 
            10.1021/jacs.0c12375. Epub 2021 Apr 23.
  PUBMED    33890764
REFERENCE   2  (bases 1 to 6497)
  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"; doi: 
            "10.1021/jacs.0c12375"; journalName: "J Am Chem Soc"; date: 
            "2021-05-5- 5"; volume: "143"; issue: "17"; pages: "6434-6446"
COMMENT     Sequence Label: pCMV-PB1-AG-FRB-TEVuncs-mTagBFP2-HA
FEATURES             Location/Qualifiers
     source          1..6497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             158..835
                     /codon_start=1
                     /product="humanized monomeric Azami-Green fluorescent 
                     protein"
                     /label=hmAzami-Green
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGVNFPPNGPVMQKKTLKWEPSTEKMYVRDGVLKGDVNMALL
                     LEGGGHYRCDFKTTYKAKKDVRLPDYHFVDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             887..1165
                     /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             1271..1981
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             1994..2020
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2154..2275
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2191..2210)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2245..2264
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2282..2737)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2419..2440)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2631..2650
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2764..2868
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2870..3227
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2870..3061
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2893..2913)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3078..3213
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3261..4055
                     /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             3261..4055
                     /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(3315..3334)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3925..3944
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4243..4262)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4287..4334
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4663..5251
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5152..5171
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5426..5729
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5730..5933
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5884..5904
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      5965..5974
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 tggcgctgga gggtctgccg gatctggagc cggtggttct gccggctccg gcgctggtgg
       61 gagcgcttcc ggaagtagtc ccggtaattc cgctgacggc ggcggaggat cgggtggtag
      121 tggtggttca ggaggaggat cgacccaagg aggatccatg gtgagcgtga tcaagcccga
      181 gatgaagatc aagctgtgca tgaggggcac cgtgaacggc cacaacttcg tgatcgaggg
      241 cgagggcaag ggcaacccct acgagggcac ccagatcctg gacctgaacg tgaccgaggg
      301 cgcccccctg cccttcgcct acgacatcct gaccaccgtg ttccagtacg gcaacagggc
      361 cttcaccaag taccccgccg acatccagga ctacttcaag cagaccttcc ccgagggcta
      421 ccactgggag aggagcatga cctacgagga ccagggcatc tgcaccgcca ccagcaacat
      481 cagcatgagg ggcgactgct tcttctacga catcaggttc gacggcgtga acttcccccc
      541 caacggcccc gtgatgcaga agaagaccct gaagtgggag cccagcaccg agaagatgta
      601 cgtgagggac ggcgtgctga agggcgacgt gaacatggcc ctgctgctgg agggcggcgg
      661 ccactacagg tgcgacttca agaccaccta caaggccaag aaggacgtga ggctgcccga
      721 ctaccacttc gtggaccaca ggatcgagat cctgaagcac gacaaggact acaacaaggt
      781 gaagctgtac gagaacgccg tggccaggta cagcatgctg cccagccagg ccaagggtac
      841 cggaactgca gcagagaatt cgggaaactc gagaacaaag cttatgatcc tctggcatga
      901 gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa ggaacgtgaa
      961 aggcatgttt gaggtgctgg agcccttgca tgctatgatg gaacggggcc cccagactct
     1021 gaaggaaaca tcctttaatc aggcctatgg tcgagattta atggaggccc aagagtggtg
     1081 caggaagtac atgaaatcag ggaatgtcaa ggacctcctc caagcctggg acctctatta
     1141 tcatgtgttc cgacgaatct caaagcccgg gggtagtggt gctggctctg gtgctggtag
     1201 tggcgctggt aacgaatatc tgcagtttct ggtcgatggt ggtagtgctg gtggttccgg
     1261 aggtgtcgac atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct
     1321 gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa
     1381 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
     1441 cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac
     1501 ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt
     1561 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     1621 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat
     1681 gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg
     1741 cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa
     1801 cgccaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta
     1861 ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca
     1921 gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa
     1981 tagatccggc tcttacccat acgatgttcc agattacgct agatcttaaa aagcggccgc
     2041 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     2101 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     2161 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     2221 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg
     2281 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     2341 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     2401 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2461 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2521 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2581 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2641 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2701 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     2761 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2821 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     2881 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2941 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3001 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3061 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3121 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     3181 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat catcaagaga
     3241 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3301 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3361 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3421 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
     3481 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3541 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3601 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3661 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     3721 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     3781 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     3841 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     3901 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     3961 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4021 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     4081 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     4141 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4201 tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac
     4261 aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt
     4321 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
     4381 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
     4441 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
     4501 ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4561 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4621 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4681 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4741 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4801 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4861 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4921 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4981 aacggggggt tcgtgcacac agcccagttt ggagcgaacg acctacaccg aactgagata
     5041 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5101 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5161 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5221 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5281 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5341 ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag
     5401 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     5461 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     5521 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     5581 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     5641 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     5701 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     5761 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     5821 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     5881 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
     5941 tgaaccgtca gatccgctag cattgccacc atggcgtcgc tcaccgtgaa ggcctacctt
     6001 ctgggcaagg aggacgcggc gcgcgagatt cgccgcttca gcttctgttg cagccccgag
     6061 cctgaggcgg aagccgaggc tgcggcgggt ccgggaccct gcgagcggct gctgagccgg
     6121 gtggccgccc tgttccccgc gctgcggcct ggcggcttcc aggcgcacta ccgcgatgag
     6181 gacggggact tggttgcctt ttccagtgac gaggaattga caatggccat gtcctacgtg
     6241 aaggatgaca tcttccgaat ctacattaaa gagaaaaccg gttctgggag tggcggtagt
     6301 ggtgctggag gcagcgcagg ctctggagca ggcggcagtg ccggaagtgg cgctggaggg
     6361 tctgccggat ctggagccgg tggttctgcc ggctccggcg ctggtgggag cgcttccgga
     6421 agtagtcccg gttctgggag tggcggtagt ggtgctggag gcagcgcagg ctctggagca
     6481 ggcggcagtg ccggaag
//
