Part:BBa_K4410006

H1-Kan^R-H2

This part is the Kan^R gene (BBa_K4410003) with the upstream and downstream 1000bp homologous fragments of argR (BBa_K4410000 and BBa_K4410001). It is the donor DNA necessary for replacing the ArgR gene in the EcN1917 genome with the KanR gene using the tool plasmid pKD46 and the λ-Red homologous recombination system. Replacing argR with Kan^R gene in EcN1917 eliminates the feedback repression of L-arginine synthesis and achieve the purpose of efficient screening.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1951
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 305
  Illegal NgoMIV site found at 2615
  Illegal NgoMIV site found at 2898
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2464
  Illegal SapI.rc site found at 2674
  

Results

Through colony PCR, we got donor DNA sequence. The result of gel electrophoresis conformed to the expected length of the donor DNA, which was about 2570bp (Figure 1). Figure 2 showed the transformation of EcN1917 with the donor DNA was successful.

Figure 1: Gel electrophoresis results of donor DNA (line1-3) gel (2570bp)

Figure 2: Plate of Donor DNA transformed EcN1917 bacteria