Difference between revisions of "Part:BBa K1638007:Design"
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===References=== | ===References=== | ||
Latest revision as of 12:38, 18 September 2015
GFP reporter with flexible linker
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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