LOCUS       sequence-493101-        6348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of synthetic Kemp eliminase (Des27.7).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6348)
  AUTHORS   Listov D, Vos E, Hoffka G, Hoch SY, Berg A, Hamer-Rogotner S, Dym O,
            Kamerlin SCL, Fleishman SJ
  TITLE     Complete computational design of high-efficiency Kemp elimination 
            enzymes.
  JOURNAL   Nature. 2025 Jul;643(8074):1421-1427. doi: 
            10.1038/s41586-025-09136-2. Epub 2025 Jun 18.
  PUBMED    40533551
REFERENCE   2  (bases 1 to 6348)
  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/s41586-025-09136-2"; journalName: "Nature"; date: 
            "2025-07"; volume: "643"; issue: "8074"; pages: "1421-1427"
COMMENT     Sequence Label: pET28-hisSumo-KE-Des27.7
FEATURES             Location/Qualifiers
     source          1..6348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..27
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     RBS             1229..1251
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1266..1290
                     /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     complement(1290..1309)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1291..1309)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1393..1412)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1618..1695
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1696..2778
                     /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(1715..1734)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3587..3778
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3772..3794)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3880..4019
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3935..3954
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4034..4051)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4205..4793)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4285..4304)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4904..4923)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4915..5730
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4915..5730
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4988..5007)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5822..6277)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5959..5980)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6171..6190
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(join(6314..6348,1..13))
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
       61 tgttagcagc cggatctcat tattagccca ccagttcgcg aatcgctttt tccggatccg
      121 gcgcacgcat aagactctca ccgattacga ctgcgtcgaa taaaccacgc atggacttta
      181 actcttcctt cgtactgtaa ccaccgattg ctaagaggac acccgtgaac ccacgctcac
      241 gggctaaacg acctaaacgc tcggctgtat tacggtcgac ctcgaacgtc ttaaggtctt
      301 gatttacgat accgatgatc tctgctcctg ccttgagggc acgctcgagc tcctcttccg
      361 tgtgaacgtc aacgatgaca cccatattta acgagtgtgc aactgctgca agttgacggt
      421 attgctcgtc gtccaataca ctcaagatca atacgatggc gtcggcttgg taagcacggg
      481 ccaagtagat ttggtagggg tctacgatga agtcgaaaca gataacgggg atgtcgactg
      541 ccgaacggat cttctctaag tcctcgtacg aaccattgtg gtacttctcg tccgtgataa
      601 caacgagggc tactgcgtac ttctcggctg tcttggcgta ctcgattggg tcacgctcta
      661 cgtcgagtcc tgaaggtgac ttacgaaggt agactgcgat gatcccgaca ccaggggcac
      721 gcaatgctgc caatacgtca cgtggtggtg gggcacgtgc ggcacgctcc ttgatctcgt
      781 cgaaaggtac gacagcctca cgtgcggcaa catcttcgcg cacatccgcc acaatcgcat
      841 ccagcgcgct cggcatgcca ccagtctgat gaagcattgc gtcgatttcg tcgccatctt
      901 ccatctccag ttcatccggg gtctgttccg cacgcaggcg acgaccatca aacaggaaag
      961 caattgcggt catgtctaca gactgacggt cacagtatgc gttcatcagc tttttcagct
     1021 gggtagaacg tttgatgcgg aaaaagactt cgttgccgtc ttgacctttc actttcaggt
     1081 tgatatgggc accgccacct tcgccacctg ccggtttctt atcttcttcg ccacccgcag
     1141 cagatccgga atggtgatga ctgcctgaat gatggtggtg tccggtatgg tgatggcctg
     1201 aatgatggtg atgcttgctc atggtatatc tccttcttaa agttaaacaa aattatttct
     1261 agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga
     1321 tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg
     1381 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
     1441 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
     1501 tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
     1561 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac
     1621 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     1681 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     1741 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     1801 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     1861 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     1921 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     1981 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     2041 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     2101 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     2161 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     2221 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     2281 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     2341 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     2401 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     2461 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
     2521 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     2581 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     2641 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     2701 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     2761 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     2821 gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg
     2881 gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca
     2941 ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat
     3001 gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac
     3061 tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc
     3121 acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct
     3181 tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa
     3241 aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct
     3301 ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg
     3361 ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt
     3421 gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag
     3481 taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg
     3541 tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga
     3601 aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa
     3661 actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc
     3721 tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     3781 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     3841 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     3901 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     3961 gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     4021 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4081 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4141 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4201 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4261 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4321 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4381 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4441 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4501 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4561 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4621 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     4681 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4741 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4801 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4861 tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     4921 catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat
     4981 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga
     5041 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     5101 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     5161 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     5221 gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     5281 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     5341 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     5401 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     5461 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     5521 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     5581 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     5641 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     5701 tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga
     5761 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5821 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     5881 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     5941 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     6001 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     6061 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     6121 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     6181 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     6241 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccaat ccggatatag
     6301 ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaa
//
