Part:BBa_K3878001

Toehold_miR141_1

This part enables the expression of the variable part of the toehold switch (Fig.1) targeting human microRNA-141-5p (hsa-miRNA-141-5p) via T7 RNA polymerase activity. It is composed of the BglII restriction site at its 5’ end and BamHI restriction site at its 3’ end. The sequence encoding for the variable part of the toehold switch is preceded by the T7 promoter. To produce a complete toehold switch, this part has to be assembled in a plasmid, directly followed by a repressed gene and the T7 terminator.

Figure 1: Scheme of toehold switch with the variable part composed of the toehold region and the hairpin. Created with BioRender.

Usage and Biology

This part is flanked by BglII and BamHI restriction sites for assembly in a receiving vector via molecular cloning using BglII and BamHI restriction enzymes as described below (Fig. 2). To produce a fully working toehold switch, this part has to be inserted in a plasmid and followed by the repressed gene and the T7 terminator, such as the Ba_K3878000.

Figure 2. Principle of the insertion of the variable part of the toehold switch in a plasmid. Created with BioRender.

This part, once cloned into a plasmid such as Ba_K3878000 and when expressed in a cell or cell-free system containing T7 RNA polymerase, will produce a functional toehold switch targeting hsa-miR-141-5p. A random nucleotide sequence has been designed between the BglII restriction sites and the T7 promoter to lengthen the size of the part. It enables easy visualization on an agarose gel. E. coli BL21 DE3 strain can be used to express this part in vivo.

This part has not been characterized yet. It is one candidate of the bank of toehold switches targeting hsa-miR-141-5p ( Ba_K3878001, Ba_K3878008, Ba_K3878009, Ba_K3878010, Ba_K3878011, Ba_K3878012) designed with the SwitchMi Designer.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 106
  Illegal SpeI site found at 142
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 142
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1
  Illegal BamHI site found at 270
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 106
  Illegal SpeI site found at 142
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 106
  Illegal SpeI site found at 142
  
• 1000
  COMPATIBLE WITH RFC[1000]