LOCUS       sequence-345292-        7089 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression and purification, mixed with polySH3 to form 
            condensates.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7089)
  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 7089)
  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: polyPRM5
FEATURES             Location/Qualifiers
     source          1..7089
                     /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             1759..1779
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1780..1797
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1810..1827)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1810..1826)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1819..1841)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2005..2022)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2175..2261
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2353..2380
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2399..2490
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2491..2559,2560..3351)
                     /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(2709..2728)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3393..3906)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3588..3609)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3800..3819
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4017..4605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4506..4525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4759..4776
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4882..4901)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5042..5064
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5058..5249)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        5745..5822
                     /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             5823..6905
                     /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(5842..5861)
                     /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 gggccatatg
     1381 aaaggaggca gctggggggg tagcaagaaa aagaaaaccg caccaacccc gcctaaacgt
     1441 agcggcggct caggtggttc tgggggtagc ggaggaagta agaaaaagaa aaccgcacca
     1501 accccgccta aacgtagcgg cggctcaggt ggttctgggg gtagcggagg aagtaagaaa
     1561 aagaaaaccg caccaacccc gcctaaacgt agcggcggct caggtggttc tgggggtagc
     1621 ggaggaagta agaaaaagaa aaccgcacca accccgccta aacgtagcgg cggctcaggt
     1681 ggttctgggg gtagcggagg aagtaagaaa aagaaaaccg caccaacccc gcctaaacgt
     1741 agcggcggct caggatccga aaacctgtat tttcagggcc atcatcatca tcatcactga
     1801 aagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     1861 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     1921 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcagc ttggctgttt
     1981 tggcggatga gataagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct
     2041 gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     2101 ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg
     2161 gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     2221 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     2281 acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
     2341 atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt
     2401 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2461 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     2521 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     2581 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     2641 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt tcccaatgat gagcactttt
     2701 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
     2761 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     2821 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     2881 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     2941 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3001 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3061 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3121 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3181 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     3241 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3301 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3361 caagtttact catatatact ttagattgat ttaccccggt tgataatcag aaaagcccca
     3421 aaaacaggaa gattgtataa gcaaatattt aaattgtaaa cgttaatatt ttgttaaaat
     3481 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     3541 tcccttataa atcaaaagaa tagcccgaga tagggttgag tgttgttcca gtttggaaca
     3601 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     3661 gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttggggtcg aggtgccgta
     3721 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     3781 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     3841 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     3901 gcgcgtaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3961 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4021 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4081 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4141 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4201 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4261 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4321 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4381 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4441 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4501 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4561 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4621 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4681 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4741 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     4801 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tgtggtgcac tctcagtaca
     4861 ccgcatgtgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4921 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4981 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5041 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     5101 cggtaaagct catcagcgtg gtcgtgcagc gattcacaga tgtctgcctg ttcatccgcg
     5161 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     5221 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     5281 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     5341 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     5401 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     5461 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     5521 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     5581 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     5641 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     5701 atcatgcgca cccgtggcca ggacccaacg ctgcccgaaa ttccgacacc atcgaatggt
     5761 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     5821 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     5881 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     5941 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     6001 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     6061 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     6121 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     6181 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     6241 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     6301 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     6361 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     6421 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     6481 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     6541 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     6601 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     6661 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     6721 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     6781 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     6841 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     6901 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacaatt ctcatgtttg
     6961 acagcttatc atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag
     7021 ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact
     7081 cccgttctg
//
