Part:BBa_K5353066

1356 LentiV-hNOS2/Flag: Puro

After purify the plasmid DNA through midi-prep, we utilize them to package the lentivirus together with DVPR and VSV-G. We utilize these lentivirus to infect HEK293T cell and the successfully infected cells will express the hNOS2 gene with Flag-tag. The hNOS2 will be used to trigger the release of the intracellular calcium. The Flag-tag will be used for detection. The lentivirus gene sequence could be integrated into about HEK293T cell genome by utilizing Puromycin (by the Puro resistance gene).

The plasmid information.

The designed DNA was constructed and we used the midi-prep preparation kit to conduct the plasmid isolation and purification of the plasmid DNA (Table 1). The inserts in each construct were further validated by the PCR reaction (Figure 1. and Figure 2).

Table 1.DNA concentrations extracted and purified.

Figure 1. Gel electrophoresis results 1.

Figure 2. Gel electrophoresis results 2.

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 4066
  Illegal BamHI site found at 608
  Illegal BamHI site found at 2913
  Illegal XhoI site found at 4465
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2340
  Illegal NgoMIV site found at 4200
  Illegal AgeI site found at 1352
  Illegal AgeI site found at 3578
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1518
  Illegal SapI site found at 951
  Illegal SapI site found at 1824
  Illegal SapI.rc site found at 2554