Part:BBa_K3039008

SP_lamB-PETase R280A

Usage and Biology

The native predicted signal peptide (Met1-Ala33) was removed from the WT PETase sequence (Seo et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The 25 AA lamB signal peptide, which allows for excretion of the enzyme via the Sec-dependent translocation pathway (Seo et al 2019)* was added to the N-terminal followed by a 13 AA His-tag. The entire amino acid sequence was codon optimised for E. coli by IDT’s on-line Codon Optimisation tool ensuring that there were no forbidden restriction sites, BsaI or SapI, to allow for TypeIIS assembly. The iGEM TypeIIS prefix and suffix were added and DNA was synthesised by IDT as a double stranded g-block. TypeIIS assembly was used to clone the resulting CDS with the T7-promoter and B0015 terminator into a high-copy number, ampicillin vector, pX1900 (University of Exeter).

Sequence and Features

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 AgeI site found at 540
  
• 1000
  COMPATIBLE WITH RFC[1000]