LOCUS       sequence-345503-        7090 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression and purification, E2 enzyme (UBC9) for 
            SUMOylation, can be recruited to FRB with rapamycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7090)
  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 7090)
  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: CyPet-FKBP-E2
FEATURES             Location/Qualifiers
     source          1..7090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     956..975
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(995..1072)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1278..1297
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1381..1400
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1381..1399
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1400..1424
                     /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             1439..1461
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1474..1503
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1507..1527
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1837..1856)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2184..2205
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(2254..2256,2257..2574)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKL"
     CDS             3139..3159
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3202..3219
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3272..3290)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3286..3333
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3370..3825
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3457..3476)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3667..3688
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3851..3955
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3956..4024,4025..4816)
                     /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(4174..4193)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4987..5575
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5476..5495
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5729..5746
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5761..5900
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5826..5845)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5986..6008
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6002..6193)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7002..7090,1..994))
                     /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"
ORIGIN
        1 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
       61 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
      121 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
      181 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
      241 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
      301 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
      361 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
      421 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
      481 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
      541 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
      601 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
      661 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
      721 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
      781 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
      841 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
      901 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
      961 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     1021 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     1081 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     1141 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     1201 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     1261 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     1321 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     1381 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
     1441 tgtttaactt taagaaggag atataccatg ggccatcacc atcaccatca ccatcaccat
     1501 cacggcgaaa acctatactt ccagggcgaa ttcgtgagca agggcgagga gctgttcggc
     1561 gggattgtgc ccatcctggt cgagctggaa ggcgacgtaa acggccacaa gttctcggta
     1621 tccggggagg gagagggaga cgctacctat gggaaactta cattaaaatt tatttgcacg
     1681 acagggaagc tgccggtgcc ttggcctact ctggttacca ccctgacatg gggcgtccaa
     1741 tgtttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgtgatgccc
     1801 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1861 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1921 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactatat cagccataac
     1981 gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcaa ggctcgccac
     2041 aacatcaccg acgggtcggt tcaactggcc gaccactatc agcaaaatac acctattggc
     2101 gacggccccg tgattctgcc cgacaaccac tacctgagca cccagtccgc gctgagcaaa
     2161 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2221 actcatggca tggacgagct gtacaagcaa ttgatggggg tacaagttga aacaattagc
     2281 cctggggacg gacgtacttt cccaaaacgc ggtcagacct gtgtggtcca ctatactggg
     2341 atgcttgagg acggtaagaa attcgactca agtcgtgacc gtaacaaacc cttcaagttc
     2401 atgttaggta aacaggaagt gattcgtggg tgggaggaag gagtggccca gatgtcagta
     2461 ggccaacgtg ctaagttgac aatttcgccc gactacgcct acggagcaac gggtcatccg
     2521 ggaattattc caccacacgc aacattagta ttcgacgtcg agttgttgaa attgaacgag
     2581 ggaggtagtg gcggatcagg tgggagtgga gggtctctga gaagccgtgc tcaggcttcg
     2641 aactctgcag ttgacggcac catgtcgggg atcgccctca gcagactcgc ccaggagagg
     2701 aaagcatgga ggaaagacca cccatttggt ttcgtggctg tcccaacaaa aaatcccgat
     2761 ggcacgatga acctcatgaa ctgggagtgc gccattccag gaaagaaagg gactccgtgg
     2821 gaaggaggct tgtttaaact acggatgctt ttcaaagatg attatccatc ttcgccacca
     2881 aaatgtaaat tcgaaccacc attatttcac ccgaatgtgt acccttcggg gacagtgtgc
     2941 ctgtccatct tagaggagga caaggactgg aggccagcca tcacaatcaa acagatccta
     3001 ttaggaatac aggaacttct aaatgaacca aatatccaag acccagctca agcagaggcc
     3061 tacacgattt actgccaaaa cagagtggag tacgagaaaa gggtccgagc acaagccaag
     3121 aagtttgcgc cctcactcga gaacctgtat tttcagggcc gtaaacgtaa acgtaaacgt
     3181 aaacgtaaag gctgagtcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     3241 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     3301 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     3361 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3421 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3481 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3541 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3601 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3661 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3721 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3781 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     3841 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3901 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3961 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4021 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4081 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4141 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4201 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4261 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4321 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4381 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4441 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     4501 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4561 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4621 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4681 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4741 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4801 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4861 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4921 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4981 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5041 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5101 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     5161 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5221 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5281 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5341 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5401 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5461 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5521 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5581 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5641 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5701 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     5761 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     5821 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     5881 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5941 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     6001 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     6061 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     6121 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     6181 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     6241 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     6301 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     6361 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     6421 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     6481 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     6541 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     6601 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     6661 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     6721 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     6781 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     6841 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     6901 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     6961 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     7021 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     7081 tgcgtattgg
//
