Part:BBa_K2591014:Design
TCF-TATA-GFP
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 56
Illegal BamHI site found at 42
Illegal XhoI site found at 52 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 120
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
BBa_K2591000
We used primers TCF_GFP_1st_F(gtaagatcaaaggtgtagagggtatataatggatccgg) and TCF_GFP_1st_R(tcctagcagaagcacagggtgcagg) to amplify the GFP part, add TATA box and remove the PstI digestion site in GFP. And then we did an extension PCR with primers TCF_GFP_2nd_F(ATTCGCGGCCGCTTCTAGAGgatcaaagggggtaagatcaaaggtgtagaggg) and TCF_GFP_2nd_R(TGCAGCGGCCGCTACTAGTActacacattgatcctagcagaagcacag) to add the prefix and suffix. At the same time, we used primers suffix_F(TACTAGTAGCGGCCGCTGCAG) and prefix_R(CTCTAGAAGCGGCCGCGAATTC) to do a reverse PCR for pSB1C3 backbone. Finally, we ligated the PCR linearized pSB1C3 and GFP part fragment by Gibson Assembly.
Source
BBa_K2591000
TCF and TATA box are both from synthesis, because they are known sequence and relatively short. GFP sequence is from our host lab.