Part:BBa_K4585011

pcDNA3.1(+)-GnRH-C

We obtained this plasmid by enzyme cutting and linking for intracellular calcium flow assay. This plasmid can be transformed into and amplified in E. coli and then used as a template for amplification. This plasmid was detected to be able to normally express GnRH-C protein in HEK 293T cells.

pcDNA3.1(+)-GnRH-C

The full-length GnRH gene sequence was purchased and the DNA sequence corresponding to the GnRH-C GAP (GnRH-associated peptide) was excised with restriction enzyme to obtain a truncated GnRH-C. Then we obtained the pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid through homologous recombination of the GnRH-C recombination insert (BBa_K4585000) with pcDNA3.1(+)-GnRH-C linearized vector (BBa_K4585016). The homologous recombination plasmid product was identified as the target product by sequencing and enzyme cutting and agarose gel electrophoresis.

1 Pattern Diagram

Fig.1 The model diagram of pcDNA3.1(+)-GnRH-C

2.1 Method

We transfected pcDNA3.1 (+) -GnRH-C plasmid into HEK 293T cells and cultured for sufficient time, after the transfected HEK 293T cells secreted the corresponding protein into the supernatant, took the supernatant of the cell culture medium for the detection of intracellular calcium flow changes, so as to obtain the secretion of GnRH protein in HEK 293T cells.

2.2 Results

The results of the detection of changes in intracellular calcium flow corresponding to the supernatant were compared with the standard curve to obtain the corresponding data on the curve to successfully prove the successful expression of pcDNA3.1 (+) -GnRH-C plasmid in HEK 293T cells.

3 Caution

After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 249
  Illegal PstI site found at 2330
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 895
  Illegal SpeI site found at 249
  Illegal PstI site found at 2330
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 12
  Illegal XhoI site found at 1000
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 249
  Illegal PstI site found at 2330
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 249
  Illegal PstI site found at 2330
  Illegal NgoMIV site found at 1440
  Illegal NgoMIV site found at 2781
  Illegal NgoMIV site found at 3064
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 882
  Illegal BsaI.rc site found at 4589
  Illegal SapI site found at 3509
  Illegal SapI.rc site found at 2630
  Illegal SapI.rc site found at 2840