Difference between revisions of "Part:BBa K1088052:Design"
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===Design Notes=== | ===Design Notes=== | ||
The linker was added according to step 1-4 as specified on the "Design" page of the flexible linker | The linker was added according to step 1-4 as specified on the "Design" page of the flexible linker | ||
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Using standard RFC[10] assembly of two parts creates scarsites (tactag). Transcription of the scarsite encodes: "uac uag" of which uag is a stop codon that would render the linker useless. Thus, standard assembly can not be employed for fusing proteins/domains. | Using standard RFC[10] assembly of two parts creates scarsites (tactag). Transcription of the scarsite encodes: "uac uag" of which uag is a stop codon that would render the linker useless. Thus, standard assembly can not be employed for fusing proteins/domains. | ||
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1) Design primers for amplification of the N-terminal protein/domain that includes BamHI-site | 1) Design primers for amplification of the N-terminal protein/domain that includes BamHI-site | ||
Latest revision as of 22:10, 16 March 2015
GFP reporter with flexible linker at N-terminus for creation of GFP fusions
- 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
The linker was added according to step 1-4 as specified on the "Design" page of the flexible linker
Using standard RFC[10] assembly of two parts creates scarsites (tactag). Transcription of the scarsite encodes: "uac uag" of which uag is a stop codon that would render the linker useless. Thus, standard assembly can not be employed for fusing proteins/domains.
1) Design primers for amplification of the N-terminal protein/domain that includes BamHI-site
a) Forward primer: 5'-cgctTCTAGAgNNN...NNN-3' - includes XbaI-site and sequence complementary to DNA sequence of N-terminal protein/domain
b) Reverse primer: 5'-atatGGATCCNNN...NNN-3' - includes BamHI-site and sequence complementary to DNA sequence of N-terminal protein/domain (OBS! do NOT include stop-codons of coding sequences)
2) PCR amplify BioBrick with designed primer
3) digest pSB1C3-Linker:GFP and PCR product with XbaI and BamHI
4) ligate the digested pSB1C3-Linker:GFP and PCR product to create a brick with your protein/domain fused with GFP
Source
BBa_I13401 and BBa_K1088051