LOCUS       sequence-493093-        6348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of synthetic Kemp eliminase (Des27.9).
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-Des27.9
FEATURES             Location/Qualifiers
     source          1..6348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             992..1014
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1029..1053
                     /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(1053..1072)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1054..1072)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1156..1175)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1381..1458
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1459..2541
                     /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(1478..1497)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3350..3541
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3535..3557)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3643..3782
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3698..3717
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3797..3814)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3968..4556)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4048..4067)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4667..4686)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4678..5493
                     /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             4678..5493
                     /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(4751..4770)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5585..6040)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5722..5743)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5934..5953
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6077..6124)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6120..6138
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 cacgggcaag acgaccgaga cgctcggccg tattacggtc aacctcgaaa gtcttcaagt
       61 cttgatttac gataccgatg atctcggccc cggccttcaa ggcacgctct aactcctctt
      121 ctgtgtgtac gtcaacgata acacccatat tcaaactgtg tgcgactgcg gccaattgac
      181 ggtattgctc gtcgtctaaa acagataaga tgaggacgat agcgtctgct tggtaagcac
      241 gtgccaagta gatttggtat gggtcaacga tgaagtcgaa acagataacg gggatgtcaa
      301 cggccgaacg gatcttctca aggtcctcgt acgaaccatt gaagtacttc tcgtcagtga
      361 agacgacgag agctacggcg tacttctcag ctgtcttggc gtactcgatt gggtcacgct
      421 caacgtcaag cccactaggg gacttacgga gatataaggc gatgataccg actccgggag
      481 cacgaagtgc ggcgagaacg tcacgtggtg gcggtgcacg tgctgcacgc tccttgatct
      541 cgtcgaaagg aacaacagcc tcacgggcag ctacatcttc gcgtacatcc gccacaatcg
      601 catccagcgc gctcggcatg ccaccagtct gatgaagcat tgcgtcgatt tcgtcgccat
      661 cttccatctc cagttcatcc ggggtctgtt ccgcacgcag gcgacgacca tcaaacagga
      721 aagcaattgc ggtcatgtct acagactgac ggtcacagta tgcgttcatc agctttttca
      781 gctgggtaga acgtttgatg cggaaaaaga cttcgttgcc gtcttgacct ttcactttca
      841 ggttgatatg ggcaccgcca ccttcgccac ctgccggttt cttatcttct tcgccacccg
      901 cagcagatcc ggaatggtga tgactgcctg aatgatggtg gtgtccggta tggtgatggc
      961 ctgaatgatg gtgatgcttg ctcatggtat atctccttct taaagttaaa caaaattatt
     1021 tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
     1081 ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag
     1141 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
     1201 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
     1261 tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca
     1321 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg
     1381 gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     1441 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     1501 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     1561 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     1621 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     1681 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     1741 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     1801 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     1861 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     1921 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     1981 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     2041 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     2101 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     2161 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     2221 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     2281 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     2341 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     2401 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     2461 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     2521 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc
     2581 accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc
     2641 gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg
     2701 acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac
     2761 gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt
     2821 cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc
     2881 cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg
     2941 ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg
     3001 caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag
     3061 tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg
     3121 ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg
     3181 agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc
     3241 cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat
     3301 cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca
     3361 ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga
     3421 gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca
     3481 cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     3541 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     3601 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     3661 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     3721 agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     3781 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3841 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3901 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3961 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4021 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4081 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4141 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4201 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4261 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4321 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4381 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4441 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4501 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4561 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4621 ttttggtcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     4681 agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct
     4741 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     4801 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     4861 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     4921 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     4981 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     5041 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     5101 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     5161 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     5221 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     5281 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     5341 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     5401 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     5461 cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt
     5521 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5581 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5641 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5701 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5761 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5821 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5881 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5941 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     6001 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc aatccggata
     6061 tagttcctcc tttcagcaaa aaacccctca agacccgttt agaggcccca aggggttatg
     6121 ctagttattg ctcagcggtg gcagcagcca actcagcttc ctttcgggct ttgttagcag
     6181 ccggatctca ttattagccc accagttcgc gaatcgcttt ttccggatcc ggcgcgcgca
     6241 tcaggctttc gccaattaag acggcgtcga aaagcccacg catcgactta agctcttcct
     6301 ttgtactgta accgccgatg gcgagtaaga caccagtgaa accacgct
//
