Part:BBa_K4449009:Design

Design

Here since Fim H1 will be cloned between the Nco1 and Xho 1 , in order to get rid of the N terminal His tag , thrombin site and the T7 tag and retain the C terminal His tag with the expressed Fim H1 protein .This is because C terminal His tag allows proper protein folding and this will ensure non interference of our aptamer binding to the thrombin site and T7 tag while performing binding assays. While PCR amplifying the insert , A single T base was inserted at the 13 th base of the forward primer sequence while designing primers so as to maintain the reading frame of the sequence during cloning in expression vector Pet28b+. The ATG start codon was deleted from the coding sequence insert of the Fim H1 since the ATG start codon is already present in the restriction enzyme recognition site ( Nco 1)which will be added in the primers.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 3738
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4074
  Illegal PstI site found at 3738
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 3738
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 3738
  Illegal NgoMIV site found at 126
  Illegal AgeI site found at 3896
  
• 1000
  COMPATIBLE WITH RFC[1000]