Part:BBa_K4585013

pGL4.35-3XHA-9XGAL4UAS-KRAB-NLS

The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid, which could express GAL4-KRAB, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). KRAB is a transcription factor that represses downstream gene expression when combined with GAL4.

pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS

The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid was obtained through homologous recombination of the KRAB homologous recombination insert (BBa_K4585003) with pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector (BBa_K4585008). 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 pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS

2.1 Method

The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid could express GAL4-KRAB, and GAL4-KRAB could bind 9×UAS and inhibit downstream gene expression, therefore GAL4-KRAB could inhibit GnRH expression. Considering that the plasmid itself contains a 9×UAS sequence, GAL4-KRAB can also suppress its own expression.

2.2 Results

HEK 293T cells were transiently transfected with GAL-VP64 and GAL-KRAB plasmids, and an appropriate amount of Luciferase plasmids were transfected to simulate GnRH. The experiment showed that the GAL-VP64 plasmid could initiate the expression of GAL4-KRAB and Luciferase.

Fig 2. Bioluminescence intensity when GAL4-VP64=GAL4-KRAB=400 ng

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 EcoRI site found at 177
  Illegal XbaI site found at 138
  Illegal XbaI site found at 216
  Illegal SpeI site found at 4980
  Illegal PstI site found at 182
  Illegal PstI site found at 1611
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 177
  Illegal SpeI site found at 4980
  Illegal PstI site found at 182
  Illegal PstI site found at 1611
  Illegal NotI site found at 203
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 177
  Illegal BglII site found at 4743
  Illegal XhoI site found at 210
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 177
  Illegal XbaI site found at 138
  Illegal XbaI site found at 216
  Illegal SpeI site found at 4980
  Illegal PstI site found at 182
  Illegal PstI site found at 1611
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 177
  Illegal XbaI site found at 138
  Illegal XbaI site found at 216
  Illegal SpeI site found at 4980
  Illegal PstI site found at 182
  Illegal PstI site found at 1611
  Illegal NgoMIV site found at 721
  Illegal NgoMIV site found at 2062
  Illegal NgoMIV site found at 2347
  Illegal AgeI site found at 274
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3875
  Illegal BsaI.rc site found at 5613
  Illegal SapI site found at 2792
  Illegal SapI.rc site found at 1911
  Illegal SapI.rc site found at 2121