Difference between revisions of "Part:BBa K1638002:Design"

(→‎Design Notes)
(→‎Design Notes)
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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) 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'
 
1.2) Forward primer: 5'-ATATGGATCCNNN...NNN-3'
 +
 
1.3) Reverse primer: 5'-ATATCTGCAGCGGCCGCTACTAGTANNN..NNN-3'
 
1.3) Reverse primer: 5'-ATATCTGCAGCGGCCGCTACTAGTANNN..NNN-3'
 +
 
2) Amplify through PCR with designed primers
 
2) Amplify through PCR with designed primers
 +
 
3) Digest PCR-product and pSB1C3 with BamHI and PstI.
 
3) Digest PCR-product and pSB1C3 with BamHI and PstI.
 +
 
4) Ligate the two digested product.
 
4) Ligate the two digested product.
  

Revision as of 14:09, 20 July 2015


T25 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 682
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 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 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 with BamHI and PstI.

4) Ligate the two digested product.

Source

asd

References