Difference between revisions of "Part:BBa K1638005:Design"

 
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
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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.  
  
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Instead we recommend the following assembly method:
  
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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.
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1.2) Forward primer: 5'-ATATGGATCCNNN...NNN-3'
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1.3) Reverse primer: 5'-ATATCTGCAGCGGCCGCTACTAGTANNN..NNN-3'
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2) Amplify through PCR with designed primers
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3) Digest PCR-product and pSB1C3-T18 with BamHI and PstI.
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4) Ligate the two digested product.
  
 
===Source===
 
===Source===

Revision as of 15:36, 20 July 2015


T18 domain of cyaA from Bordetella pertussis (IPTG inducible)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 711
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 252
    Illegal NgoMIV site found at 662
    Illegal AgeI site found at 468
  • 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

..

References