LOCUS       sequence-494350-        6345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of synthetic Kemp eliminase (Des61).
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-Des61
FEATURES             Location/Qualifiers
     source          1..6345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(53..71)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      67..114
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      151..606
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(238..257)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     448..469
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(698..1513)
                     /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(698..1513)
                     /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     1421..1440
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1505..1524
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1635..2223
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2124..2143
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2377..2394
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2409..2548
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2474..2493)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2634..2656
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2650..2841)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3650..4732)
                     /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     4694..4713
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4733..4810)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5016..5035
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5119..5138
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5119..5137
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5138..5162
                     /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             5177..5199
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5596..5607
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
       61 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
      121 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
      181 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      241 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      301 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      361 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      421 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      481 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
      541 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
      601 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
      661 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
      721 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
      781 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
      841 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
      901 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
      961 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     1021 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     1081 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     1141 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     1201 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     1261 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     1321 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     1381 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     1441 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     1501 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     1561 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1621 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1681 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1741 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     1801 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1861 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     1921 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1981 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2041 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2101 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     2161 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     2221 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     2281 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     2341 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     2401 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     2461 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     2521 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     2581 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     2641 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     2701 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     2761 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     2821 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     2881 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     2941 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     3001 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     3061 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     3121 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     3181 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     3241 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     3301 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     3361 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     3421 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     3481 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     3541 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     3601 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     3661 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3721 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     3781 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     3841 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     3901 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     3961 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     4021 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     4081 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     4141 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     4201 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     4261 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     4321 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     4381 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     4441 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     4501 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     4561 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     4621 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     4681 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     4741 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     4801 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     4861 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     4921 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     4981 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     5041 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     5101 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
     5161 cccctctaga aataattttg tttaacttta agaaggagat ataccatgag caagcatcac
     5221 catcattcag gccatcacca taccggacac caccatcatt caggcagtca tcaccattcc
     5281 ggatctgctg cgggtggcga agaagataag aaaccggcag gtggcgaagg tggcggtgcc
     5341 catatcaacc tgaaagtgaa aggtcaagac ggcaacgaag tctttttccg catcaaacgt
     5401 tctacccagc tgaaaaagct gatgaacgca tactgtgacc gtcagtctgt agacatgacc
     5461 gcaattgctt tcctgtttga tggtcgtcgc ctgcgtgcgg aacagacccc ggatgaactg
     5521 gagatggaag atggcgacga aatcgacgca atgcttcatc agactggtgg catggatcgc
     5581 gcggatctgc gcaacattga aggccgcctc gcagagattg ccaagacgat cgctgccgac
     5641 ttacaaccta agcccttacc accaccacca cctggtcgta atttcgaggc agcattacgt
     5701 cgtcctggga tcgcccttat cctcgcatgt ttaaaggcca cacccgctaa gggtttatta
     5761 cgtgaggact tcgacccagt acgtatcgct gctatctacc gtcactacgc agacgctatc
     5821 gccgtcatca cggacgagaa gtacttacaa ggttcgatgt ccttcttacg tcaagttcgt
     5881 gccgttgcac ctcaacctat cctctgtgtc gacttcatcg tagacccctc gcaaatcgac
     5941 gaggcacgtg cagcaggagc tgacgccatc ctcttaatcg tagccatcct cacgcctgag
     6001 gcactcaaga agctctacga gtacgccaag tccttaggtt tagaccccat cgtagaggtt
     6061 cacactgagg aagagctcaa gcgtgccctt aagctcggtg cacgtatcat cggtatctta
     6121 aatatcgact tgcgtacagg gactatcgac cgtaatgtag cagagcacct catgcgttta
     6181 gcccgtaagc aagggttccg tggtgtatta atcgctatcg gtggatactc tactaaggaa
     6241 gagctcaaga gtttagaggg gttagccgac gccgtcctta tctgggaagc catcatgacg
     6301 gcacccgaca tcgcaaaaaa actggcgaaa ctgcgcggct aataa
//
