Reporter

Part:BBa_K1638007:Design

Designed by: Jens Sivkr Pettersen   Group: iGEM15_SDU-Denmark   (2015-05-10)
Revision as of 12:38, 18 September 2015 by Jpettersen (Talk | contribs) (→‎Source)

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GFP reporter with flexible linker


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 674


Design Notes

Standard assembly [RFC10] with this part creates scarsite (uacuag). Scarsites encodes stopcodons, which is inconvenient for the right use of this GFP reporter. The GFP reporter contains a 5'-end BamHI restriction site to be used in fusion to proteins with a 3'-end BamHI restriction site. For implementation, we recommend the following assembly method:

5) Design primers for amplification of N-terminal fusion protein domain that includes a 3'-end BamHI res. site.

5.1) Forward: 5'-CGCTTCTAGAGNNN...NNN-3' (includes XbaI res. site)

5.2) Reverse: 5'-ATATGGATCCNNN...NNN-3' (includes BamHI res. site)

6) Run PCR with designed primers.

7) Digest PCR-products and plasmid containing GFP reporter with BamHI and PstI(or SpeI) res. site.

8) Ligate backbone PCR product

Source

BBa_E0040

References