Part:BBa_K3815013

Part of the EPH1 gene of Ipomoea nil to synthesize dsRNA for RNAi
This is a section of the EPH1 gene of Ipomoea nil to synthesize dsRNA for RNAi. To product dsRNA, this sequence is inserted in the L4440 plasmid, and transformed into HT115(DE3).

purpose

This is a gene for a transcription factor that positively regulates PCD in Ipomoea nil. Silencing this gene is expected to extend the flowering time of Ipomoea nil.[1]

Usage and Biology

RNAi
RNAi (RNA interference) is a process in which externally introduced dsRNA suppresses the expression of genes that have complementary sequences to the dsRNA.

L4440
L4440 is a plasmid vector having two convergent T7 promoters adjacent to the multi-cloning site. By inserting a portion of the target gene sequence into the multi-cloning site of this plasmid, the target sequence is transcribed from both sides, and dsRNA can be obtained when both parts are annealed.

HT115(DE3)
HT115 (DE3) is an RNase III-deficient E. coli strain that has been modified to express T7 RNA polymerase from an IPTG-inducible promoter. It lacks dsRNA-specific RNase III, which allows it to produce high levels of specific dsRNA. These attributes allow HT115 (DE3) to be a promising strain for the preparation of large amounts of viral dsRNA in vivo.

Sequence and features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 9
  
• 1000
  COMPATIBLE WITH RFC[1000]

cloning

This part was inserted in L4440. The L4440 has two t7 promoters, and this ​part is transcribed from both sides.

Reference

1 Shibuya, K., Shimizu, K., Niki, T., and Ichimura, K. (2014). Identification of a NAC transcription factor, EPHEMERAL1, that controls petal senescence in Japanese morning glory. Plant J. 79, 1044–1051.