Coding
T18 domain

Part:BBa_K1638004:Design

Designed by: Jens Sivkr Pettersen   Group: iGEM15_SDU-Denmark   (2015-05-10)
Revision as of 15:30, 20 July 2015 by Jpettersen (Talk | contribs) (→‎Design Notes)


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-T25 with BamHI and PstI.

4) Ligate the two digested product.

Source

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References