Part:BBa_K2591014:Design

TCF-TATA-GFP

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 56
  Illegal BamHI site found at 42
  Illegal XhoI site found at 52
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 120
  
• 1000
  COMPATIBLE 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