LOCUS       sequence-333353-        1563 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses AviTag-DogCatcher-MBP in bacterial cytoplasm. The AviTag 
            enables site-specific biotinylation by BirA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1563)
  AUTHORS   Keeble AH, Yadav VK, Ferla MP, Bauer CC, Chuntharpursat-Bon E, Huang
            J, Bon RS, Howarth M
  TITLE     DogCatcher allows loop-friendly protein-protein ligation.
  JOURNAL   Cell Chem Biol. 2021 Jul 28. pii: S2451-9456(21)00315-9. doi: 
            10.1016/j.chembiol.2021.07.005.
  PUBMED    34324879
REFERENCE   2  (bases 1 to 1563)
  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: "Cell Chem 
            Biol. 2021 Jul 28. pii: S2451-9456(21)00315-9. doi: 
            10.1016/j.chembiol.2021.07.005."
COMMENT     Sequence Label: pET28-AviTag-DogCatcher-MBP
FEATURES             Location/Qualifiers
     source          1..1563
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             13..30
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             40..57
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             64..108
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             121..432
                     /codon_start=1
                     /product="engineered fragment of the RrgA adhesin of 
                     Streptococcus pneumoniae"
                     /label=DogCatcher
                     /note="forms a covalent bond with an intra-domain protein 
                     loop containing the DogTag peptide (Keeble et al., 2022)"
                     /translation="KLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTY
                     QDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ
                     "
     CDS             451..1548
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1522..1545
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
ORIGIN
        1 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat
       61 atgggtctga atgatatttt cgaagcgcag aaaattgaat ggcatgaagg tagcggaatg
      121 aaactgggcg agattgaatt tattaaagtg gacaaaaccg ataaaaagcc gctgcgtggt
      181 gccgtgttta gcctgcagaa acagcatccc gactatcccg atatctatgg cgcgattgat
      241 cagaatggga cctatcaaga tgtgcgtacc ggcgaagatg gtaaactgac ctttacgaat
      301 ctgagcgatg gcaaatatcg cctgattgaa aatagcgaac ccccgggcta taaaccggtg
      361 cagaataagc cgattgtgag ctttcgtatt gtggatggcg aagtgcgtga tgtgaccagt
      421 attgtgccgc agggtagtgg tgaaagtggt aaaatcgaag aaggtaaact ggtaatctgg
      481 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
      541 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
      601 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
      661 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
      721 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
      781 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
      841 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
      901 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
      961 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     1021 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     1081 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     1141 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     1201 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     1261 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     1321 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     1381 ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     1441 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     1501 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     1561 taa
//
