Part:BBa_K1442115

B2 3

Each unique RNA dependent RNA polymerase (RdRP) initiates de novo replication of a RNA strand by interacting with RdRP-specific RNA sequences, henceforth called RdRP/RNA promoters. The RdRP chosen for our project is taken from the Hepatitis C virus (HCV). The B2 RNA promoter was identified as such by Heinz, Kao (2000) , along with a number of other sequences derived from the Brome Mosaic Virus (BMV). All of them possess a few common characteristics: an initiation cytidylate at the 3’ end, where the replication starts; and a stable secondary structure – single stranded tail and a stem of various length. Replication stops at the 5’ end. Please refer to Section: RdRP-directed Replication for further details.

This is derived from the 3’ end of the Cytomegalovirus RNA3 minus strand. It contains a non-templated guanylate at the 3’ end. These are genomic plus-strand RNAs that can direct BMV minus-strand initiation. This has a 3’ single stranded tail which is required for efficient RNA synthesis.

Usage

Used as an RNA promoter to direct replication by RdRP.

RdRP-directed Replication

1. The DNA strand gets transcribed and then translated, whereby the RdRP protein is produced.

2. The active RdRP binds to the 3’ end of the transcribed plus –sense RNA strand. It produces its complementary minus-sense RNA strand.

3. The RdRP interacts with the 5’ end of the replicated minus-sense RNA strand. It produces the plus-sense RNA strand again and completes the full replication of the RNA. The new RNA strand can act as a template as well and the process of replication can start over.

Mathematical Modelling

E-coli

Where c, α-,β are constants, E stands for the RdRP, G – for GFP. R- is amount of minus-sense RNA strands, R+ amount of plus-sense RNA strands; µ- is degradation rate of the minus-sense strands and µ+of the plus-sense.

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 38
  Illegal NgoMIV site found at 67
  
• 1000
  COMPATIBLE WITH RFC[1000]