LOCUS       sequence-345502-        7081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression and purification, E2 enzyme (UBC9) for 
            SUMOylation, can be recruited to FRB with rapamycin, CyPet acts as a
            FRET donor to Ypet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7081)
  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 7081)
  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: mCherry-FKBP-E2
FEATURES             Location/Qualifiers
     source          1..7081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(778..1860)
                     /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     1822..1841
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1861..1938)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2144..2163
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2247..2266
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2247..2265
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2266..2290
                     /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)."
     RBS             2305..2327
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2340..2369
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2373..2393
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2400..3104
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2542..2560)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2810..2829
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3004..3027
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             join(3111..3113,3114..3431)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKL"
     CDS             3996..4016
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4059..4076
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4129..4147)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4143..4190
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4227..4682
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4314..4333)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4524..4545
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4708..4812
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4813..4881,4882..5673)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5031..5050)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5844..6432
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6333..6352
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6586..6603
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6618..6757
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6683..6702)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6843..6865
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6859..7050)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
       61 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      121 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
      181 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
      241 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
      301 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
      361 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
      421 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
      481 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
      541 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
      601 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
      661 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
      721 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
      781 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
      841 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
      901 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
      961 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1021 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     1081 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1141 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1201 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     1261 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     1321 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     1381 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     1441 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     1501 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     1561 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     1621 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     1681 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     1741 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     1801 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     1861 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1921 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1981 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     2041 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     2101 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     2161 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     2221 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     2281 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggcc
     2341 atcaccatca ccatcaccat caccatcacg gcgaaaacct atacttccag ggcgaattcg
     2401 tgagcaaggg cgaggaggac aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     2461 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     2521 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg
     2581 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     2641 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     2701 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     2761 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     2821 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     2881 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgccgagg
     2941 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     3001 tcaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgcg
     3061 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagcaattg atgggggtac
     3121 aagttgaaac aattagccct ggggacggac gtactttccc aaaacgcggt cagacctgtg
     3181 tggtccacta tactgggatg cttgaggacg gtaagaaatt cgactcaagt cgtgaccgta
     3241 acaaaccctt caagttcatg ttaggtaaac aggaagtgat tcgtgggtgg gaggaaggag
     3301 tggcccagat gtcagtaggc caacgtgcta agttgacaat ttcgcccgac tacgcctacg
     3361 gagcaacggg tcatccggga attattccac cacacgcaac attagtattc gacgtcgagt
     3421 tgttgaaatt gaacgaggga ggtagtggcg gatcaggtgg gagtggaggg tctctgagaa
     3481 gccgtgctca ggcttcgaac tctgcagttg acggcaccat gtcggggatc gccctcagca
     3541 gactcgccca ggagaggaaa gcatggagga aagaccaccc atttggtttc gtggctgtcc
     3601 caacaaaaaa tcccgatggc acgatgaacc tcatgaactg ggagtgcgcc attccaggaa
     3661 agaaagggac tccgtgggaa ggaggcttgt ttaaactacg gatgcttttc aaagatgatt
     3721 atccatcttc gccaccaaaa tgtaaattcg aaccaccatt atttcacccg aatgtgtacc
     3781 cttcggggac agtgtgcctg tccatcttag aggaggacaa ggactggagg ccagccatca
     3841 caatcaaaca gatcctatta ggaatacagg aacttctaaa tgaaccaaat atccaagacc
     3901 cagctcaagc agaggcctac acgatttact gccaaaacag agtggagtac gagaaaaggg
     3961 tccgagcaca agccaagaag tttgcgccct cactcgagaa cctgtatttt cagggccgta
     4021 aacgtaaacg taaacgtaaa cgtaaaggct gagtcgagca ccaccaccac caccactgag
     4081 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     4141 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     4201 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     4261 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     4321 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4381 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4441 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     4501 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     4561 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4621 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     4681 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4741 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4801 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4861 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4921 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4981 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5041 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5101 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5161 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5221 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5281 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5341 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5401 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5461 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5521 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5581 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5641 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5701 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5761 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5821 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5881 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5941 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     6001 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6061 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6121 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6181 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6241 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6301 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6361 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6421 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6481 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6541 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6601 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     6661 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     6721 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     6781 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     6841 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     6901 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     6961 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     7021 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     7081 c
//
