LOCUS       sequence-355800-        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-TEVcs-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-TEVcs-mTagBFP2-HA
FEATURES             Location/Qualifiers
     source          1..6497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      540..549
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1230..1907
                     /codon_start=1
                     /product="humanized monomeric Azami-Green fluorescent 
                     protein"
                     /label=hmAzami-Green
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGVNFPPNGPVMQKKTLKWEPSTEKMYVRDGVLKGDVNMALL
                     LEGGGHYRCDFKTTYKAKKDVRLPDYHFVDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             1959..2237
                     /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             2343..3053
                     /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             3066..3092
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3226..3347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3263..3282)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3317..3336
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3354..3809)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3491..3512)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3703..3722
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3836..3940
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3942..4299
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3942..4133
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3965..3985)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4150..4285
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4333..5127
                     /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             4333..5127
                     /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(4387..4406)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4997..5016
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5315..5334)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5359..5406
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5735..6323
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6224..6243
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcattg
      541 ccaccatggc gtcgctcacc gtgaaggcct accttctggg caaggaggac gcggcgcgcg
      601 agattcgccg cttcagcttc tgttgcagcc ccgagcctga ggcggaagcc gaggctgcgg
      661 cgggtccggg accctgcgag cggctgctga gccgggtggc cgccctgttc cccgcgctgc
      721 ggcctggcgg cttccaggcg cactaccgcg atgaggacgg ggacttggtt gccttttcca
      781 gtgacgagga attgacaatg gccatgtcct acgtgaagga tgacatcttc cgaatctaca
      841 ttaaagagaa aaccggttct gggagtggcg gtagtggtgc tggaggcagc gcaggctctg
      901 gagcaggcgg cagtgccgga agtggcgctg gagggtctgc cggatctgga gccggtggtt
      961 ctgccggctc cggcgctggt gggagcgctt ccggaagtag tcccggttct gggagtggcg
     1021 gtagtggtgc tggaggcagc gcaggctctg gagcaggcgg cagtgccgga agtggcgctg
     1081 gagggtctgc cggatctgga gccggtggtt ctgccggctc cggcgctggt gggagcgctt
     1141 ccggaagtag tcccggtaat tccgctgacg gcggcggagg atcgggtggt agtggtggtt
     1201 caggaggagg atcgacccaa ggaggatcca tggtgagcgt gatcaagccc gagatgaaga
     1261 tcaagctgtg catgaggggc accgtgaacg gccacaactt cgtgatcgag ggcgagggca
     1321 agggcaaccc ctacgagggc acccagatcc tggacctgaa cgtgaccgag ggcgcccccc
     1381 tgcccttcgc ctacgacatc ctgaccaccg tgttccagta cggcaacagg gccttcacca
     1441 agtaccccgc cgacatccag gactacttca agcagacctt ccccgagggc taccactggg
     1501 agaggagcat gacctacgag gaccagggca tctgcaccgc caccagcaac atcagcatga
     1561 ggggcgactg cttcttctac gacatcaggt tcgacggcgt gaacttcccc cccaacggcc
     1621 ccgtgatgca gaagaagacc ctgaagtggg agcccagcac cgagaagatg tacgtgaggg
     1681 acggcgtgct gaagggcgac gtgaacatgg ccctgctgct ggagggcggc ggccactaca
     1741 ggtgcgactt caagaccacc tacaaggcca agaaggacgt gaggctgccc gactaccact
     1801 tcgtggacca caggatcgag atcctgaagc acgacaagga ctacaacaag gtgaagctgt
     1861 acgagaacgc cgtggccagg tacagcatgc tgcccagcca ggccaagggt accggaactg
     1921 cagcagagaa ttcgggaaac tcgagaacaa agcttatgat cctctggcat gagatgtggc
     1981 atgaaggcct ggaagaggca tctcgtttgt actttgggga aaggaacgtg aaaggcatgt
     2041 ttgaggtgct ggagcccttg catgctatga tggaacgggg cccccagact ctgaaggaaa
     2101 catcctttaa tcaggcctat ggtcgagatt taatggaggc ccaagagtgg tgcaggaagt
     2161 acatgaaatc agggaatgtc aaggacctcc tccaagcctg ggacctctat tatcatgtgt
     2221 tccgacgaat ctcaaagccc gggggtagtg gtgctggctc tggtgctggt agtggcgctg
     2281 gtgaaaacct gtattttcag ctggtcgatg gtggtagtgc tggtggttcc ggaggtgtcg
     2341 acatggtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag ctgtacatgg
     2401 agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg
     2461 agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg
     2521 acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca
     2581 tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat
     2641 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
     2701 tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga
     2761 aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag
     2821 gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca aacgccaaga
     2881 ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg
     2941 actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg
     3001 tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt aatagatccg
     3061 gctcttaccc atacgatgtt ccagattacg ctagatctta aaaagcggcc gcgactctag
     3121 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     3181 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     3241 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3301 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt
     3361 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3421 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     3481 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3541 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3601 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3661 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3721 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3781 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg
     3841 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3901 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag
     3961 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     4021 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     4081 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     4141 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4201 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4261 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcatcaaga gacaggatga
     4321 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4381 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4441 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4501 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4561 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4621 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4681 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4741 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4801 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4861 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4921 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4981 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5041 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     5101 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     5161 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     5221 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     5281 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     5341 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     5401 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     5461 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     5521 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     5581 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5641 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5701 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5761 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5821 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5881 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5941 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6001 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6061 gttcgtgcac acagcccagt ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6121 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6181 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6241 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6301 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6361 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6421 gtattaccgc catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc
     6481 ccatatatgg agttccg
//
