Part:BBa_K1638004:Design

T18 domain of CyaA from Bordetella pertussis

Assembly Compatibility:

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

  Illegal BamHI site found at 550
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 91
  Illegal NgoMIV site found at 501
  Illegal AgeI site found at 307
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Addition of a protein coding domain to the suffix by Standard Assembly RFC[10] creates a scar-site. As the scarsite encodes a stop codon (uacuag), this will leave the fused protein coding domain untranslated.

Instead we recommend the following assembly method:

1) Design primers for amplification of the C-terminal fusion protein with BamHI resitriction site included in the forward primer. The primers must also include prefix and suffix.

1.2) Forward primer: 5'-ATATGGATCCNNN...NNN-3'

1.3) Reverse primer: 5'-ATATCTGCAGCGGCCGCTACTAGTANNN..NNN-3'

2) Amplify through PCR with designed primers

3) Digest PCR-product and pSB1C3-T18 with BamHI and PstI.

4) Ligate the two digested product.

Source

pUT18C