LOCUS       sequence-443737-        6713 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses asparaginyl endopeptidase butelase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6713)
  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 6713)
  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: pET28-Butelase
FEATURES             Location/Qualifiers
     source          1..6713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(122..169)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     165..183
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(236..253)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1687..1704)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1714..1731)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1750..1772
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1787..1811
                     /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(1811..1830)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1812..1830)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1914..1933)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2139..2216
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2217..3299
                     /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(2236..2255)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4108..4299
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4293..4315)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4401..4540
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4456..4475
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4555..4572)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4726..5314)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4806..4825)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5425..5444)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5436..6251
                     /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             5436..6251
                     /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(5509..5528)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(6343..6713,1..85))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6480..6501)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6692..6711
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
       61 cttaatgcgc cgctacaggg cgcgtcccat tcgccaatcc ggatatagtt cctcctttca
      121 gcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctagt tattgctcag
      181 cggtggcagc agccaactca gcttcctttc gggctttgtt agcagccgga tctcagtggt
      241 ggtggtggtg gtgctcgagc taggtagata cttggttata agaatgatta aaagctttta
      301 aacgctaaaa ccggcatgca ggcttgacca cggattatcc ggaatgctaa cacatgcctg
      361 tgcgcttgct tccgccattt gttctttgcg aatacctgca ttacacagat tggcaaagct
      421 acgcatgtgt ttcataccat attcgctcag gctaccacaa tgggtttcaa aggtacgaat
      481 cagggttttg aaacaatccc aatcatcaac aaccggcaga cctgccggac gaacattatt
      541 cagcatttta tgacctttct cgataccaaa cagcagtttg ccaatatgtt tcacgctgtt
      601 atcaatatgc atacgatgtg ccataacttc acgcagctgt ttttttgctt cggctttacg
      661 gctgctacct tccggtgcac gacgatattt ttcccaataa tgaatcagat ctgcatcacg
      721 ctggctaact gctttacgcg gtgtacccag gctgttttta tcggtaaagg tattgttatc
      781 atttgcagga tcggtgccca tataaacaaa cagggtctgt ttgctcagac caacatcacc
      841 atactgaaca acatgggtgc catcttcaat cagggccaca atggttttgt ttttcacaac
      901 ttcatactgc tgctgaaagg tttcggtctg cagattatga acatcacaat cttccagcca
      961 tgcaacgcta aacaggtcac caacacaaac atcatattcc ggaggcggac tcggatgctg
     1021 cagcggacaa taggtaaccc agctgctttc accggtatcg cttgcaccca tcacataaat
     1081 gttatgatct tccggcagca gaccatcaaa catgctgccg ctttcgcagc tttcaacata
     1141 aaacacaatg cttttatagg tgccgcttgc atgttttttt ttcagcacgt cattcagatc
     1201 gcttgctgca atatacggtt tgctcggcat acccagaaca ccggcaccac catgatcggt
     1261 ataatagatg aacacgtgat cattcggacc gctatccaga actttaccgc taccggtgcc
     1321 ggtcagtgcg cttttatttg ccagcagaac tgcataaaag ttaggcggat taatatcttc
     1381 gcccacataa tctttcggaa cacctttata cacatcggaa ccatacggat gattgataat
     1441 aacacccgga tgcggattgc tttcattgta ggcgatatca tcatacataa acacgatgat
     1501 gttttcgtct ttcaggccac cttttttcag aatctgatat gcatgacaaa catcggcctg
     1561 atgacgataa ttaacataac ctttgctacc tgcaaccaga actgcccaac gggtgccttc
     1621 aacattatca tctgcctgga aaaatttgct tgcctggctc ggcagacgca gaaaatcatc
     1681 acggatggat ccgcgcggca ccaggccgct gctgtgatga tgatgatgat ggctgctgcg
     1741 catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg
     1801 ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta
     1861 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
     1921 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
     1981 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
     2041 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
     2101 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa
     2161 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     2221 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     2281 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     2341 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     2401 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     2461 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     2521 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     2581 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     2641 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     2701 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     2761 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     2821 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     2881 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     2941 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     3001 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     3061 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     3121 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     3181 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     3241 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     3301 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc
     3361 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     3421 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg
     3481 gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg
     3541 gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt
     3601 ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg
     3661 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     3721 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     3781 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     3841 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     3901 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     3961 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     4021 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     4081 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     4141 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     4201 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc
     4261 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4321 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     4381 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     4441 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat tgcggtgtga
     4501 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
     4561 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4621 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4681 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4741 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4801 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4861 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4921 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4981 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5041 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5101 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5161 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5221 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5281 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5341 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac
     5401 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     5461 cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     5521 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg
     5581 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     5641 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     5701 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg
     5761 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     5821 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     5881 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     5941 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     6001 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     6061 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     6121 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     6181 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     6241 agtttttcta agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa
     6301 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     6361 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     6421 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     6481 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     6541 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     6601 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     6661 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcg
//
