LOCUS       sequence-146162-        7739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  create protein aggregates in living cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7739)
  AUTHORS   Miyazaki Y, Mizumoto K, Dey G, Kudo T, Perrino J, Chen LC, Meyer T, 
            Wandless TJ
  TITLE     A method to rapidly create protein aggregates in living cells.
  JOURNAL   Nat Commun. 2016 May 27;7:11689. doi: 10.1038/ncomms11689.
  PUBMED    27229621
REFERENCE   2  (bases 1 to 7739)
  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 
            Commun."; date: "2016-05-27"; volume: "7:11689. doi"; pages: " 
            10.1038/ncomms11689"
COMMENT     Sequence Label: AgDD
FEATURES             Location/Qualifiers
     source          1..7739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             46..366
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KPE"
     CDS             373..1086
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             373..1083
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(400..428)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1023..1043
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    1090..1211
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1127..1146)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1181..1200
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   1973..2007
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2498..2514)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2498..2514)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2511..2533)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2522..2538
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2546..2552,2553..2570,2571..2576))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2591..2612
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2729..2746)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2900..3488)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2980..2999)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3659..4450,4451..4519))
                     /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     4282..4301
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4520..4624)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4650..5105)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4787..4808)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4999..5018
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5232..5254
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5246..5263
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5247..5263
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   5393..5455
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     misc_feature    5887..6003
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        6025..6404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6407..6682
                     /label=chicken beta-actin promoter
     intron          6683..7691
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     7615..7637
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     7699..7718
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
ORIGIN
        1 gaattcgcca ccatgctggc cctgaagctg gctggtctag atattggagt gcaggtggaa
       61 accatctccc caggagacgg gcgcaccttc cccaagcgcg gccagacctg tgtggtgcac
      121 tacaccggga tgcttgaaga tggaaagaaa gtcgattcct cccgggacag aaacaagccc
      181 tttaagttta tgctaggcaa gcaggaggtg atccgaggct gggaagaagg ggttgcccag
      241 atgagtgtgg gtcagagagc caaactgact atatctccag attatgccta tggtgccact
      301 gggcacccag gcatcatccc accacatgcc actctcgtct tcgatgtgga gcttctaaaa
      361 ccggaagaat tcagcaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa
      421 ttagatggtg atgttaatgg gcacaaattt tctgtccgtg gagagggtga aggtgatgct
      481 acaaacggaa aactcaccct taaatttatt tgcactactg gaaaactacc tgttccgtgg
      541 ccaacacttg tcactactct gacctatggt gttcaatgct tttcccgtta tccggatcac
      601 atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaacgcact
      661 atatctttca aagatgacgg gacctacaag acgcgtgctg aagtcaagtt tgaaggtgat
      721 acccttgtta atcgtatcga gttaaagggt attgatttta aagaagatgg aaacattctt
      781 ggacacaaac tcgagtacaa ctttaactca cacaatgtat acatcacggc agacaaacaa
      841 aagaatggaa tcaaagctaa cttcaaaatt cgccacaacg ttgaagatgg ttccgttcaa
      901 ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac
      961 aaccattacc tgtcgacaca atctgtcctt tcgaaagatc ccaacgaaaa gcgtgaccac
     1021 atggtccttc ttgagtttgt aactgctgct gggattacac atggcatgga tgagctctac
     1081 aaataagtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     1141 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     1201 aatgtatctt atcatgtctg gaattgactc aaatgatgtc aattagtcta tcagaagcta
     1261 tctggtctcc cttccggggg acaagacatc cctgtttaat atttaaacag cagtgttccc
     1321 aaactgggtt cttatatccc ttgctctggt caaccaggtt gcagggtttc ctgtcctcac
     1381 aggaacgaag tccctaaaga aacagtggca gccaggttta gccccggaat tgactggatt
     1441 ccttttttag ggcccattgg tatggctttt tccccgtatc cccccaggtg tctgcaggct
     1501 caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg
     1561 gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc
     1621 ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt
     1681 tggggggggg ctgtccctga tatctataac aagaaaatat atatataata agttatcacg
     1741 taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta
     1801 aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact
     1861 taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg
     1921 cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc
     1981 aattttacgc agactatctt tctagggtta atctagctgc atcaggatca tatcgtcggg
     2041 tcttttttcc ggctcagtca tcgcccaagc tggcgctatc tgggcatcgg ggaggaagaa
     2101 gcccgtgcct tttcccgcga ggttgaagcg gcatggaaag agtttgccga ggatgactgc
     2161 tgctgcattg acgttgagcg aaaacgcacg tttaccatga tgattcggga aggtgtggcc
     2221 atgcacgcct ttaacggtga actgttcgtt caggccacct gggataccag ttcgtcgcgg
     2281 cttttccgga cacagttccg gatggtcagc ccgaagcgca tcagcaaccc gaacaatacc
     2341 ggcgacagcc ggaactgccg tgccggtgtg cagattaatg acagcggtgc ggcgctggga
     2401 tattacgtca gcgaggacgg gtatcctggc tggatgccgc agaaatggac atggataccc
     2461 cgtgagttac ccggcgggcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2521 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2581 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2641 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2701 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2761 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2821 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2881 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2941 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3001 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3061 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3121 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3181 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3241 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3301 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     3361 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3421 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3481 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3541 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3601 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3661 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3721 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3781 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3841 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3901 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3961 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4021 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4081 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4141 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4201 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4261 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4321 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4381 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4441 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4501 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4561 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4621 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     4681 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     4741 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     4801 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     4861 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     4921 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     4981 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     5041 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     5101 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     5161 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     5221 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cctcgttcat
     5281 tcacgttttt gaacccgtgg aggacgggca gactcgcggt gcaaatgtgt tttacagcgt
     5341 gatggagcag atgaagatgc tcgacacgct gcagaacacg cagctagatt aaccctagaa
     5401 agataatcat attgtgacgt acgttaaaga taatcatgcg taaaattgac gcatgtgttt
     5461 tatcggtctg tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat
     5521 ttacacttac atactaataa taaattcaac aaacaattta tttatgttta tttatttatt
     5581 aaaaaaaaac aaaaactcaa aatttcttct ataaagtaac aaaactttta tgagggacag
     5641 ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg
     5701 agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg
     5761 cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg
     5821 ctgctctttg agcctgcaga cacctggggg gatacgggga aaaggcctcc aaggccagac
     5881 tagaactttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     5941 cataatagca acagacatac aaacaaagaa ttacaaaaac aaattacaaa aattcaaaat
     6001 tttatcgatc acgagactag cctcgacatt gattattgac tagttattaa tagtaatcaa
     6061 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     6121 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     6181 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt
     6241 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     6301 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     6361 ctacttggca gtacatctac gtattagtca tcgctattac catgggtcga ggtgagcccc
     6421 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt
     6481 attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcgcg cgccaggcgg
     6541 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca
     6601 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
     6661 aaagcgaagc gcgcggcggg cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg
     6721 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg
     6781 gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc
     6841 ttttctgtgg ctgcgtgaaa gccttaaagg gctccgggag ggccctttgt gcggggggga
     6901 gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct
     6961 gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc
     7021 gaggggagcg cggccggggg cggtgccccg cggtgcgggg gggctgcgag gggaacaaag
     7081 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc
     7141 tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg
     7201 gggctccgtg cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg
     7261 ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc
     7321 ggccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     7381 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctggcggagc cgaaatctgg
     7441 gaggcgccgc cgcaccccct ctagcgggcg cgggcgaagc ggtgcggcgc cggcaggaag
     7501 gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccatctccag
     7561 cctcggggct gccgcagggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     7621 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
     7681 ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaa
//
