LOCUS       sequence-366720-        6950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP tagged triple RGG scaffold with N-terminal FRB. 
            Expression induced in media containing galactose..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6950)
  AUTHORS   Garabedian MV, Wang W, Dabdoub JB, Tong M, Caldwell RM, Benman W, 
            Schuster BS, Deiters A, Good MC
  TITLE     Designer membraneless organelles sequester native factors for 
            control of cell behavior.
  JOURNAL   Nat Chem Biol. 2021 Sep;17(9):998-1007. doi: 
            10.1038/s41589-021-00840-4. Epub 2021 Aug 2.
  PUBMED    34341589
REFERENCE   2  (bases 1 to 6950)
  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: 
            "10.1038/s41589-021-00840-4"; journalName: "Nat Chem Biol"; date: 
            "2021-09"; volume: "17"; issue: "9"; pages: "998-1007"
COMMENT     Sequence Label: Yiplac211 proGAL1-FRB-RGG-GFP-RGG-RGG
FEATURES             Location/Qualifiers
     source          1..6950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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)."
     primer_bind     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        243..684
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    243..360
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     655..678
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             691..960
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             1489..1509
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1510..1512,1513..1515,1516..2226)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1555..1576)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1816..1835)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2163..2184
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      3249..3436
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3304..3328)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3448..3465)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3448..3464)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3457..3479)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3673..3692)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3792..3814
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3932..4735)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4736..4951)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4958..4976)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5044..5148
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5149..5217,5218..6009)
                     /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     complement(5367..5386)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6180..6768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6669..6688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6922..6939
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcta
      241 gacggattag aagccgccga gcgggtgaca gccctccgaa ggaagactct cctccgtgcg
      301 tcctcgtctt caccggtcgc gttcctgaaa cgcagatgtg cctcgcgccg cactgctccg
      361 aacaataaag attctacaat actagctttt atggttatga agaggaaaaa ttggcagtaa
      421 cctggcccca caaaccttca aatgaacgaa tcaaattaac aaccatagga tgataatgcg
      481 attagttttt tagccttatt tctggggtaa ttaatcagcg aagcgatgat ttttgatcta
      541 ttaacagata tataaatgca aaaactgcat aaccacttta actaatactt tcaacatttt
      601 cggtttgtat tacttcttat tcaaatgtaa taaaagtatc aacaaaaaat tgttaatata
      661 cctctatact ttaacgtcaa ggaggagctc gagatgtggc atgaaggcct ggaagaggca
      721 tctcgtttgt actttgggga aaggaacgtg aaaggcatgt ttgaggtgct ggagcccttg
      781 catgctatga tggaacgggg cccccagact ctgaaggaaa catcctttaa tcaggcctat
      841 ggtcgagatt taatggaggc ccaagagtgg tgcaggaagt acatgaaatc agggaatgtc
      901 aaggacctca cccaagcctg ggacctctat tatcatgtgt tccgacgaat ctcaaagcag
      961 ggatccatgg aatcgaatca atctaacaac ggtggatcgg gaaacgcagc attgaatcgt
     1021 gggggccgtt acgtaccacc acacttgaga ggcggagatg gtggtgccgc cgctgctgcc
     1081 tcagccggag gcgacgacag acgcggtggg gccgggggtg gtggctatcg tagaggtggt
     1141 ggaaattcag ggggcggcgg cggaggaggc tacgatcgtg gatacaacga caaccgggat
     1201 gaccgtgaca atcggggcgg ctccggcggt tatggccggg acagaaatta cgaggatcgc
     1261 ggttataacg ggggtggtgg gggaggaggc aatcgcggct acaataataa ccgcggtggg
     1321 ggtggaggcg gctataatcg gcaggaccgt ggagacggcg gttctagtaa tttctcgcgg
     1381 ggagggtata acaacagaga tgagggctca gacaacagag gctcaggcag atcatacaac
     1441 aacgatcggc gcgacaatgg tggtgacggg ggcggaggcg gttccctcga gaatttatat
     1501 ttccagggca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1561 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1621 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1681 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     1741 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1801 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1861 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1921 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1981 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2041 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2101 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat
     2161 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2221 tacaaggaat ctaaccaatc aaacaatggc ggctccggta acgcggcgct taacaggggc
     2281 ggtcgttacg tgcctccgca cctgagaggg ggcgacggag gtgcggctgc tgccgcgagt
     2341 gccggtggtg atgacaggcg tggtggtgcg ggtggcgggg gctatcgtcg tggaggcggt
     2401 aattccgggg gaggtggtgg tgggggttac gacagagggt acaatgataa ccgtgatgat
     2461 cgtgacaaca gaggcgggag tggcggttac ggtagggatc gtaactatga agacagaggc
     2521 tataacgggg gtggtggtgg cggtggcaat agaggttata ataacaaccg tggcggcggt
     2581 ggaggcgggt ataacagaca ggatagaggg gacggcggca gtagcaactt ctcccgtggc
     2641 ggatataaca accgtgacga aggctctgac aataggggtt ctggtaggtc ttacaataat
     2701 gataggagag acaatggcgg cgacggtaag cttatggaat ccaatcaatc caataacggt
     2761 ggctcgggta atgccgctct gaatcgtggt ggccgctacg tgccgccgca tctgcgtggt
     2821 ggcgatggtg gtgcagcagc agctgcatct gctggcggtg atgaccgtcg cggcggtgcg
     2881 ggcggtggcg gttatcgtcg cggcggtggc aatagtggtg gcggtggcgg tggcggttat
     2941 gatcgtggtt acaatgacaa ccgtgatgac cgcgataacc gtggcggttc cggcggttac
     3001 ggtcgtgatc gtaattatga agaccgtggt tacaacggcg gtggcggtgg cggtggcaat
     3061 cgtggctata acaataaccg tggtggcggt ggcggtggct acaatcgcca ggatcgtggt
     3121 gacggtggca gctctaactt ttcgcgcggt ggctataata accgtgatga aggcagcgac
     3181 aaccgcggta gtggccgttc ctacaataac gatcgtcgcg acaatggtgg cgatggctaa
     3241 tgaccggtgc gaatttctta tgatttatga tttttattat taaataagtt ataaaaaaaa
     3301 taagtgtata caaattttaa agtgactctt aggttttaaa acgaaaattc ttattcttga
     3361 gtaactcttt cctgtaggtc aggttgcttt ctcaggtata gtatgaggtc gctcttattg
     3421 accacacctc taccggctgc agaattcact ggccgtcgtt ttacaacgtc gtgactggga
     3481 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     3541 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     3601 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     3661 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     3721 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3781 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3841 gagacgaaag ggcgggtaat aactgatata attaaattga agctctaatt tgtgagttta
     3901 gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat cttctcaaat
     3961 atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc cttccctttg
     4021 caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca tcatccacgg
     4081 ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg ggtgtcataa
     4141 tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata aagccgataa
     4201 caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct ccagtagata
     4261 gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc tttgttactt
     4321 cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg tgtgcattcg
     4381 taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat ttgactgtat
     4441 taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg gcggataatg
     4501 cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc acatgtgttt
     4561 ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc ttggtggtac
     4621 gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta aatagcttgg
     4681 cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc gacatgattt
     4741 atcttcgttt cctgcatgtt tttgttctgt gcagttgggt taagaatact gggcaatttc
     4801 atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc tccttccttc
     4861 gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac cgaaatcaaa
     4921 aaaaagaata aaaaaaaaat gatgaattga aaagctggcc tcgtgatacg cctattttta
     4981 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     5041 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5101 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5161 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5221 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5281 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5341 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5401 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5461 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5521 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5581 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5641 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5701 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5761 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5821 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5881 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5941 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6001 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6061 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6121 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6241 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6301 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6481 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6541 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6601 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6661 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6721 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6781 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6841 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6901 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
//
