LOCUS       sequence-443739-        5280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Butelase AY with Val237Ala and Thr238Tyr mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5280)
  AUTHORS   Singh AK, Antonenko A, Kocyla A, Krezel A
  TITLE     An efficient and easily obtainable butelase variant for 
            chemoenzymatic ligation and modification of peptides and proteins.
  JOURNAL   Microb Cell Fact. 2024 Nov 30;23(1):325. doi: 
            10.1186/s12934-024-02598-5.
  PUBMED    39614317
REFERENCE   2  (bases 1 to 5280)
  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"; journalName: "Microb Cell
            Fact."; date: "2024-11-30"; volume: "23(1):325. doi"; pages: " 
            10.1186/s12934-024-02598-5"
COMMENT     Sequence Label: Butelase AY
FEATURES             Location/Qualifiers
     source          1..5280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             160..204
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(242..260)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      256..303
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(533..1348)
                     /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(533..1348)
                     /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     1256..1275
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1349..1440)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1456..2205)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(1466..1483)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(1706..1725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2401..3483)
                     /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     3445..3464
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3484..3561)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3607..3626
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3607..3625
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3626..3650
                     /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)."
     CDS             3706..3723
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3730..4020
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
ORIGIN
        1 cacatgcgta gctttgccaa tctgtgtaat gcaggtattc gcaaagaaca aatggcggaa
       61 gcaagcgcac aggcatgtgt tagcattccg gataatccgt ggtcaagcct gcatgccggt
      121 tttagcgttt aaaagcttgc ggccgcactc gagtctggta aagaaaccgc tgctgcgaaa
      181 tttgaacgcc agcacatgga ctcgtctact agcgcagctt aattaaccta ggctgctgcc
      241 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      301 ttgctgaaac ctcaggcatt tgagaagcac acggtcacac tgcttccggt agtcaataaa
      361 ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga accgacgaca
      421 agctgacgac cgggtctccg caagtggcac ttttcgggga aatgtgcgcg gaacccctat
      481 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
      541 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
      601 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
      661 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
      721 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
      781 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
      841 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
      901 cgagacgaaa tacgcggtcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
      961 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     1021 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     1081 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     1141 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     1201 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     1261 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     1321 aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat tgaagcattt
     1381 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     1441 taggcatgca gcgctcttcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac
     1501 tgcggcgagc ggtgtcagct cactcaaaag cggtaatacg gttatccaca gaatcagggg
     1561 ataaagccgg aaagaacatg tgagcaaaaa gcaaagcacc ggaagaagcc aacgccgcag
     1621 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagccag
     1681 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1741 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1801 ggaagcgtgg cgctttctca tagctcacgc tgttggtatc tcagttcggt gtaggtcgtt
     1861 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1921 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1981 attggtaact gatttagagg actttgtctt gaagttatgc acctgttaag gctaaactga
     2041 aagaacagat tttggtgagt gcggtcctcc aacccactta ccttggttca aagagttggt
     2101 agctcagcga accttgagaa aaccaccgtt ggtagcggtg gtttttcttt atttatgaga
     2161 tgatgaatca atcggtctat caagtcaacg aacagctatt ccgttactct agatttcagt
     2221 gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtaatt
     2281 ctcatgttag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     2341 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     2401 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2461 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     2521 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     2581 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     2641 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     2701 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     2761 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     2821 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     2881 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     2941 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     3001 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     3061 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     3121 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     3181 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     3241 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     3301 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     3361 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     3421 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     3481 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     3541 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     3601 ggaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctgtagaaa
     3661 taattttgtt taactttaat aaggagatat accatgggca gcagccatca tcatcatcat
     3721 cacagcacta gtgactcaga agtcaatcaa gaagctaagc cagaggtcaa gccagaagtc
     3781 aagcctgaga ctcacatcaa tttaaaggtg tccgatggat cttcagaaat cttctttaag
     3841 atcaaaaaga ccactccttt aagaaggctg atggaagcgt tcgctaaaag acagggtaag
     3901 gaaatggact ccttaagatt cttgtacgac ggtattagaa ttcaagctga tcagacccct
     3961 gaagatttgg acatggagga taacgatatt attgaggctc acagagaaca gattggtgga
     4021 tccatccgtg atgattttct gcgtctgccg agccaggcaa gcaaattttt ccaggcagat
     4081 gataatgttg aaggcacccg ttgggcagtt ctggttgcag gtagcaaagg ttatgttaat
     4141 tatcgtcatc aggccgatgt ttgtcatgca tatcagattc tgaaaaaagg tggcctgaaa
     4201 gacgaaaaca tcatcgtgtt tatgtatgat gatatcgcct acaatgaaag caatccgcat
     4261 ccgggtgtta ttatcaatca tccgtatggt tccgatgtgt ataaaggtgt tccgaaagat
     4321 tatgtgggcg aagatattaa tccgcctaac ttttatgcag ttctgctggc aaataaaagc
     4381 gcactgaccg gcaccggtag cggtaaagtt ctggatagcg gtccgaatga tcacgtgttc
     4441 atctattata ccgatcatgg tggtgccggt gttctgggta tgccgagcaa accgtatatt
     4501 gcagcaagcg atctgaatga cgtgctgaaa aaaaaacatg caagcggcac ctataaaagc
     4561 attgtgtttt atgttgaaag ctgcgaaagc ggcagcatgt ttgatggtct gctgccggaa
     4621 gatcataaca tttatgtgat gggtgcaagc gataccggtg aaagcagctg ggcgtactat
     4681 tgtccgctgc agcatccgag tccgcctccg gaatatgatg tttgtgttgg tgacctgttt
     4741 agcgttgcat ggctggaaga ttgtgatgtt cataatctgc agaccgaaac ctttcagcag
     4801 cagtatgaag ttgtgaaaaa caaaaccatt gtggccctga ttgaagatgg cacccatgtt
     4861 gttcagtatg gtgatgttgg tctgagcaaa cagaccctgt ttgtttatat gggcaccgat
     4921 cctgcaaatg ataacaatac ctttaccgat aaaaacagcc tgggtacacc gcgtaaagca
     4981 gttagccagc gtgatgcaga tctgattcat tattgggaaa aatatcgtcg tgcaccggaa
     5041 ggtagcagcc gtaaagccga agcaaaaaaa cagctgcgtg aagttatggc acatcgtatg
     5101 catattgata acagcgtgaa acatattggc aaactgctgt ttggtatcga gaaaggtcat
     5161 aaaatgctga ataatgttcg tccggcaggt ctgccggttg ttgatgattg ggattgtttc
     5221 aaaaccctga ttcgtacctt tgaaacccat tgtggtagcc tgagcgaata tggtatgaaa
//
