Part:BBa_K4410005

H1-argR-H2

BBa_K4410005 is argR gene (BBa_K4410002) with its upstream and downstream 1000bp homologous fragments (BBa_K4410000 and BBa_K4410001). The H1-argR-H2 is obtained via colony PCR from EcN1917 genome. Using the λ-Red homologous recombination system, to replace the ArgR gene in the EcN1917 genome with the KanR gene, it is necessary to add the upstream and downstream 1000bp homologous fragments of ArgR, at both ends of the KanR, which is the donor DNA H1-KanR-H2 (BBa_K4410006). The H1-argR-H2 is used to construct the donor DNA by TA cloning and one-step cloning. Compared with the fusion PCR, this method has a higher success rate of constructing donor DNA.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 1033
  Illegal PstI site found at 335
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 1033
  Illegal PstI site found at 335
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 1033
  Illegal PstI site found at 335
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 1033
  Illegal PstI site found at 335
  Illegal NgoMIV site found at 279
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

After the H1-argR-H2 extraction from EcN, we did gel electrophoresis to test the length of the sequence. The result showed that the target sequence was about 1527bp as we expected (Figure 1). Gel electrophotography of recombinant pMD19T showed the length was 2680bp as expected (Figure 2).

Figure 1: Gel electrophoresis results of H1-argR-H2 (line6-8) gel (1527bp).

Figure 2: Gel electrophoresis results of H1-argR-H2 with pMD19 T Vector(2680bp).