LOCUS       sequence-345501-        6709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression and purification, E2 enzyme (UBC9) for 
            SUMOylation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6709)
  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 6709)
  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-E2
FEATURES             Location/Qualifiers
     source          1..6709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     922..941
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(961..1038)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1244..1263
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1347..1366
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1347..1365
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1366..1390
                     /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             1405..1427
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1440..1469
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1473..1493
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1500..2204
                     /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(1642..1660)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1910..1929
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2104..2127
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2724..2744
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2787..2804
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2857..2875)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2871..2918
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2955..3410
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3042..3061)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3436..3540
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3541..3609,3610..4401)
                     /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(3759..3778)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4572..5160
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5061..5080
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5314..5331
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5346..5485
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5411..5430)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5571..5593
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5587..5778)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6587..6709,1..960))
                     /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 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
       61 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
      121 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
      181 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
      241 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
      301 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
      361 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
      421 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
      481 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
      541 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
      601 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
      661 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
      721 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
      781 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
      841 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
      901 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
      961 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     1021 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     1081 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     1141 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     1201 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     1261 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     1321 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     1381 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggcc
     1441 atcaccatca ccatcaccat caccatcacg gcgaaaacct atacttccag ggcgaattcg
     1501 tgagcaaggg cgaggaggac aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     1561 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1621 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg
     1681 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     1741 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     1801 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     1861 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     1921 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     1981 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgccgagg
     2041 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     2101 tcaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgcg
     2161 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtccgga ggaggtagtg
     2221 gcggatcagg tgggagtgga gggtctatgt cggggatcgc cctcagcaga ctcgcccagg
     2281 agaggaaagc atggaggaaa gaccacccat ttggtttcgt ggctgtccca acaaaaaatc
     2341 ccgatggcac gatgaacctc atgaactggg agtgcgccat tccaggaaag aaagggactc
     2401 cgtgggaagg aggcttgttt aaaatacgga tgcttttcaa agatgattat ccatcttcgc
     2461 caccaaaatg taaattcgaa ccaccattat ttcacccgaa tgtgtaccct tcggggacag
     2521 tgtgcctgtc catcttagag gaggacaagg actggaggcc agccatcaca atcaaacaga
     2581 tcctattagg aatacaggaa cttctaaatg aaccaaatat ccaagaccca gctcaagcag
     2641 aggcctacac gatttactgc caaaacagag tggagtacga gaaaagggtc cgagcacaag
     2701 ccaagaagtt tgcgccctca ctcgagaacc tgtattttca gggccgtaaa cgtaaacgta
     2761 aacgtaaacg taaaggctga gtcgagcacc accaccacca ccactgagat ccggctgcta
     2821 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     2881 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     2941 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3001 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3061 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3121 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3181 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3241 gttctttaat agtggactct tgttccaaaa tggaacaaca ctcaacccta tctcggtcta
     3301 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3361 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     3421 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3481 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3541 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3601 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3661 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3721 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3781 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3841 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3901 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3961 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     4021 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     4081 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4141 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4201 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4261 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4321 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4381 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4441 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4501 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4561 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4621 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4681 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4741 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4801 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4861 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4921 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4981 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5041 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5101 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5161 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5221 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     5281 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     5341 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     5401 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     5461 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     5521 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5581 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     5641 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     5701 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     5761 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     5821 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     5881 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     5941 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     6001 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     6061 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     6121 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     6181 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     6241 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     6301 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     6361 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     6421 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     6481 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     6541 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     6601 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     6661 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagac
//
