LOCUS       sequence-351710-        5639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of PB1-AzamiGreen-FRB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5639)
  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 5639)
  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
FEATURES             Location/Qualifiers
     source          1..5639
                     /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"
     polyA_signal    2368..2489
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2405..2424)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2459..2478
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2496..2951)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2633..2654)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2845..2864
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2978..3082
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3084..3441
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3084..3275
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3107..3127)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3292..3427
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3475..4269
                     /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             3475..4269
                     /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(3529..3548)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4139..4158
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4457..4476)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4501..4548
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4877..5465
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5366..5385
                     /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 gggtaagcgg ccgcgactct agatcataat cagccatacc
     2281 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     2341 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     2401 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2461 ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt
     2521 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2581 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     2641 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     2701 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     2761 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     2821 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     2881 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     2941 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3001 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3061 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3121 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3181 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3241 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3301 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3361 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3421 ggaggcctag gcttttgcaa agatcatcaa gagacaggat gaggatcgtt tcgcatgatt
     3481 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3541 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3601 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     3661 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     3721 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     3781 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3841 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3901 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3961 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     4021 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4081 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4141 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4201 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4261 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4321 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4381 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4441 ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     4501 cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg
     4561 ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg
     4621 cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg
     4681 cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga
     4741 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4801 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4861 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4921 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4981 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5041 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5101 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5161 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5221 gtttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5281 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5341 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5401 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5461 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5521 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcatt
     5581 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccg
//
