Coding

Part:BBa_K200001:Design

Designed by: Royah Vaezi, Dineka Khurmi   Group: iGEM09_Imperial College London   (2009-08-12)
Revision as of 21:11, 19 October 2009 by Royah (Talk | contribs) (→‎Design Notes)

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Dam methylase -> Dam


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 306
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The Dam methylase was obtained through PCR using BL21 as the genomic DNA template and the Pfu Ultra II enzyme. Primers were designed to include overhangs coding for XbaI and SpeI recognition sites in order to allow the gene to be BioBricked according to the BioBrick Standard. The primers are as follows:

  • Forward PCR primer containing XbaI overhang (bold) for BioBricking:


GCTCTAGATGAAGAAAAATCGCGCTTTTTTG

  • Reverse PCR primer containing SpeI overhang (bold) for BioBricking:


GGACTAGTATTATTTTTTCGCGGGTGAAAC


BioBrick overhang shown in bold

Source

This is one of three site-specific DNA methylases found in most laboratory strains of E. coli.

References