LOCUS       sequence-345293-        7734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression and purification, mixed with polyPRM to form 
            condensates, can bind FKBP in the presence of rapamycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7734)
  AUTHORS   Peeples W, Rosen MK
  TITLE     Mechanistic dissection of increased enzymatic rate in a 
            phase-separated compartment.
  JOURNAL   Nat Chem Biol. 2021 Jun;17(6):693-702. doi: 
            10.1038/s41589-021-00801-x. Epub 2021 May 25.
  PUBMED    34035521
REFERENCE   2  (bases 1 to 7734)
  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/s41589-021-00801-x"; journalName: "Nat Chem Biol"; date: 
            "2021-06"; volume: "17"; issue: "6"; pages: "693-702"
COMMENT     Sequence Label: FRB-polySH33
FEATURES             Location/Qualifiers
     source          1..7734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(59..64,65..80,81..87)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    95..111
                     /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     100..122
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             181..1281
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1138..1147
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1255..1278
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1330..1341
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1354..1374
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1387..1656
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             2404..2424
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2425..2442
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2455..2472)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2455..2471)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2464..2486)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2650..2667)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2820..2906
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2998..3025
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3044..3135
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3136..3204,3205..3996)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3354..3373)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4038..4551)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4233..4254)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4445..4464
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4662..5250
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5151..5170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5404..5421
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5527..5546)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5687..5709
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5703..5894)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6390..6467
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6468..7550
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(6487..6506)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt
       61 gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca
      121 ggaaacagcc agtccgttta ggtgttttca cgagcacttc accaacaagg accatagatt
      181 atgaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt
      241 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat
      301 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt
      361 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc
      421 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac
      481 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa
      541 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg
      601 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg
      661 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa
      721 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt
      781 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa
      841 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa
      901 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt
      961 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc
     1021 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg
     1081 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc
     1141 accatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc
     1201 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa
     1261 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac
     1321 aacctcggga tcgagggaag gatttcagaa tttgaaaacc tgtattttca gggcgaattc
     1381 atgttagaga tgtggcatga aggacttgag gaagcttccc gcctttactt tggtgaacgt
     1441 aatgttaagg gcatgttcga agtattggaa cccctgcacg cgatgatgga acgtgggcca
     1501 cagacattga aagaaacaag ctttaaccaa gcttacggac gtgatctgat ggaggcgcaa
     1561 gagtggtgcc gtaagtatat gaagtccggt aacgtaaaag acctgacaca agcatgggat
     1621 ttgtactacc atgtttttcg ccgtatctcg aagcaagttg atggaggtag tggcggatca
     1681 ggtgggagtg gagggtctca tatggacctc aacatgcccg cttatgtgaa atttaactac
     1741 atggctgaga gagaggatga attatcattg ataaagggga caaaggtgat cgtcatggag
     1801 aaaagcagtg atgggtggtg gcgtggtagc tacaatggac aagttggatg gttcccttca
     1861 aactatgtaa ctgaagaagg tgacagtcct ttggcgtctg gtgcgggtgg ttctgaaggt
     1921 ggtggttctg aaggtggtac ctctggtgcg accgacctca acatgcccgc ttatgtgaaa
     1981 tttaactaca tggctgagag agaggatgaa ttatcattga taaaggggac aaaggtgatc
     2041 gtcatggaga aaagcagtga tgggtggtgg cgtggtagct acaatggaca agttggatgg
     2101 ttcccttcaa actatgtaac tgaagaaggt gacagtcctt tggcatccgg agcaggaggc
     2161 tccgaaggag gaggctccga gggaggaaca tccggagcaa cagacctcaa catgcccgct
     2221 tatgtgaaat ttaactacat ggctgagaga gaggatgaat tatcattgat aaaggggaca
     2281 aaggtgatcg tcatggagaa aagcagtgat gggtggtggc gtggtagcta caatggacaa
     2341 gttggatggt tcccttcaaa ctatgtaact gaagaaggtg acagtccttt gggaggcgga
     2401 tccgaaaacc tgtattttca gggccatcat catcatcatc actgaaagct tggcactggc
     2461 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     2521 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     2581 ccaacagttg cgcagcctga atggcgaatg gcagcttggc tgttttggcg gatgagataa
     2641 gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt
     2701 gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg
     2761 ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc
     2821 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     2881 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     2941 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga
     3001 aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt atttttctaa
     3061 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3121 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3181 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3241 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3301 gagagttttc gccccgaaga acgtttccca atgatgagca cttttaaagt tctgctatgt
     3361 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     3421 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3481 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3541 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3601 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3661 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3721 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3781 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3841 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3901 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3961 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4021 atactttaga ttgatttacc ccggttgata atcagaaaag ccccaaaaac aggaagattg
     4081 tataagcaaa tatttaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt
     4141 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     4201 aagaatagcc cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     4261 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     4321 gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     4381 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     4441 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     4501 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg taaaaggatc
     4561 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4621 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4681 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4741 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4801 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4861 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4921 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4981 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5041 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5101 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5161 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5221 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5281 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5341 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     5401 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     5461 catctgtgcg gtatttcaca ccgcatgtgg tgcactctca gtacaccgca tgtggtgcac
     5521 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     5581 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5641 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5701 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     5761 gcgtggtcgt gcagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     5821 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     5881 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     5941 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     6001 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     6061 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     6121 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     6181 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     6241 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     6301 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     6361 ggccaggacc caacgctgcc cgaaattccg acaccatcga atggtgcaaa acctttcgcg
     6421 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     6481 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     6541 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     6601 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     6661 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     6721 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     6781 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     6841 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     6901 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     6961 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     7021 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     7081 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     7141 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     7201 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     7261 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     7321 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     7381 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     7441 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     7501 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     7561 attaatgtaa gttagctcac tcattaggca caattctcat gtttgacagc ttatcatcga
     7621 ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt
     7681 gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctg
//
