Part:BBa_K1639015

VP16 Transcriptional Activation Domain with miR373 Binding Site

Usage and Biology

Cloning into pTet-off

This part contains VP16 activator complex with appropriate

Figure 1:Schematic diagram of cloning into pTet-off

enzyme cut sites and miRNA binding sites for high-miRNas. Originally it's same with VP16 part of tetracycline-controlled transactivator (tTA), comprising a fusion of the tetracycline repressor TetR with the C-terminal activation domain of herpes simplex virus VP16 By including Sal1 cut site at the beginning of this part we can change it with original tTA part found in pTet-off plasmid. (Figure 1) We performed ligation and then transformed the plasmids into BL21 bacteria. After 16 hours incubation at 37 C, we performed colony PCR and cut-check to check for positive clones. Despite trying this strategy 7 or 8 times, we were unable to successfully clone this g-block into pTET off.

Below there is a whole diagram of Cancer module. This part is first step in this machinery. (Figure 2)

Figure 2

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 335
  Illegal PstI site found at 341
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 335
  Illegal PstI site found at 341
  Illegal NotI site found at 327
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 335
  Illegal PstI site found at 341
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 335
  Illegal PstI site found at 341
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 320