Part:BBa_K3407000:Design
Reversible nonoxidative vanillate / 4-hydroxybenzoate decarboxylase from B. subtilis (bsdBCD)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE 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.