Part:BBa_K3407000:Design

Reversible nonoxidative vanillate / 4-hydroxybenzoate decarboxylase from B. subtilis (bsdBCD)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 477
  Illegal AgeI site found at 67
  Illegal AgeI site found at 319
  Illegal AgeI site found at 1148
  Illegal AgeI site found at 1238
  Illegal AgeI site found at 1440
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design notes

The sequence of the three genes (bsdBCD) was taken from Bacillus subtilis subsp. subtilis str. 168 complete genome (GenBank: AL009126.3), 412540...414822. The genes were codon-optimized for expression in Escherichia coli using the GenSmartTM Codon Optimization Tool [source]. The forbidden restriction enzymes sites are the following: BioBrick forbidden restriction sites (EcoRI, XbaI, SpeI, PstI) and Type IIS forbidden restriction sites (BsaI, SapI and Bpil). As the three genes were not in the same reading frame due to nucleotides in between them, each of them was codon-optimized separately. The expression cassette (BBaX) was synthesized in the backbone vector pTWIST_Amp_HighCopy from Twist bioscience, containing the viral promoter and terminator from the T7 bacteriophage. This synthetically synthetised plasmid is referred as pTWIST_bsdBCD.