LOCUS       sequence-495003-        6345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of synthetic Kemp eliminase (Des27.7 F113L).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6345)
  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 6345)
  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_F113L
FEATURES             Location/Qualifiers
     source          1..6345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(50..68)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      64..111
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      148..603
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(235..254)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     445..466
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(695..1510)
                     /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             complement(695..1510)
                     /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     1418..1437
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1502..1521
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1632..2220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2121..2140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2374..2391
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2406..2545
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2471..2490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2631..2653
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2647..2838)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRS*TLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3647..4729)
                     /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     4691..4710
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4730..4807)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5013..5032
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5116..5135
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5116..5134
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5135..5159
                     /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             5174..5196
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
       61 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
      121 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
      181 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
      241 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
      301 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
      361 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
      421 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
      481 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
      541 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
      601 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
      661 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
      721 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
      781 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
      841 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
      901 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
      961 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     1021 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     1081 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     1141 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     1201 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     1261 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     1321 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     1381 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     1441 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     1501 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     1561 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1621 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1681 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1741 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     1801 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1861 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1921 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1981 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2041 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     2101 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     2161 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     2221 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     2281 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     2341 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     2401 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     2461 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     2521 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     2581 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     2641 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     2701 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     2761 gagtttctcc agaagcgtta atgtctagct tctgataaag cgggccatgt taagggcggt
     2821 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     2881 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     2941 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     3001 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     3061 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     3121 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     3181 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     3241 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     3301 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     3361 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     3421 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     3481 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     3541 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     3601 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     3661 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3721 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     3781 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     3841 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     3901 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     3961 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     4021 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     4081 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     4141 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     4201 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     4261 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     4321 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     4381 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     4441 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     4501 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     4561 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     4621 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     4681 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     4741 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     4801 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     4861 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     4921 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     4981 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     5041 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     5101 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     5161 ctctagaaat aattttgttt aactttaaga aggagatata ccatgagcaa gcatcaccat
     5221 cattcaggcc atcaccatac cggacaccac catcattcag gcagtcatca ccattccgga
     5281 tctgctgcgg gtggcgaaga agataagaaa ccggcaggtg gcgaaggtgg cggtgcccat
     5341 atcaacctga aagtgaaagg tcaagacggc aacgaagtct ttttccgcat caaacgttct
     5401 acccagctga aaaagctgat gaacgcatac tgtgaccgtc agtctgtaga catgaccgca
     5461 attgctttcc tgtttgatgg tcgtcgcctg cgtgcggaac agaccccgga tgaactggag
     5521 atggaagatg gcgacgaaat cgacgcaatg cttcatcaga ctggtggcat gcccagtgct
     5581 cttgacgcca ttgtcgctga tgttcgcgag gacgttgcgg cacgtgaggc ggtcgttccc
     5641 tttgatgaga ttaaagaacg cgcagcacgt gcgcctcctc ctcgcgatgt tctggcggcg
     5701 ctccgagctc caggtgttgg cattatcgca gtctatcttc gcaaaagccc gagcggcctc
     5761 gatgtcgagc gcgatcctat agagtacgcg aagaccgccg agaagtacgc ggtcgcgctc
     5821 gttgttatca cagacgaaaa atatcacaat ggttcctatg aggatttaga gaaaatacgc
     5881 tccgcagtgg acatcccggt catttgtttg gacttcatcg tagatcctta ccagatctac
     5941 ctggcgagag cctatcaggc cgatgctatt gtattgattc tgtctgtact ggatgatgaa
     6001 cagtatcggc aactggctgc agtcgcacat tctctcaaca tgggggtcat cgtcgatgta
     6061 cacaccgagg aggagctgga acgcgccctc aaagctggag ccgaaattat tgggatcgtt
     6121 aatcaggact taaagacatt tgaagttgat agaaacaccg cggaacgtct gggtcggtta
     6181 gcacgcgaac gcgggtttac aggcgtcctt ctggccattg gcggctattc tacgaaagaa
     6241 gagcttaagt ctatgcgcgg cttatttgat gctgttgtaa ttggagagag ccttatgcgt
     6301 gcgccggacc ctgaaaaagc tattagagag ttagtgggtt aatga
//
