Part:BBa_K3179100

pTREK

This part consists of Vector pTRE, miR663b-target, 2kt-EGFP and miR885-5p-target. There is a CMV promoter on pTRE that can transcribe mRNAs containing miR663b-target, 2kt-EGFP and miR885-5p-target. The translation of this mRNA is regulated by L7Ae and the amount of intracellular free miRNA663b and miRNA885-5p. When miRNA663b, miRNA885-5p and L7Ae exist, the translation of this mRNA is suppressed. The mRNA’s translation product is EGFP, and green fluorescence can be observed to facilitate observation of gene expression results.

In our design, pTRE-miR663b-2kt-EGFP-miR885-5p is used for co-transduction with pTRE-L7Ae-miR592, and the compatibility and effectiveness of L7Ae-Kturn system and Tet-On system can be tested by measuring the expression amount of EGFP.

The 2Kt-EGFP sequence is cloned from PTK295.pTK295 was a gift from Ron Weiss (Addgene plasmid # 59564 ; http://n2t.net/addgene:59564 ; RRID:Addgene_59564)

Usage and Biology

We placed the miRNA sensor downstream of the pTRE promoter to detect if Tet on could work compatibly with it. miRNA-592 mimics were transported to switch off the miRNA sensor, while miRNA-885-5p inhibitor were transported to switch on the miRNA sensor. Experimental result shows that the two systems are well compatible.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 195
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 201
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 857