Part:BBa_K4585007

pcDNA3.1(+)-3xHA-Gal4-KRAB-NLS linearized vector

The purpose of the linear vector is to perform homologous recombination with the KRAB homologous recombination fragment to obtain the target product pcDNA3.1 (+) -3xHA-Gal 4-KRAB-NLS plasmid.

pcDNA3.1(+)-3×HA-GAL4-VP64-NLS

We based on the sequence of the pcDNA3.1 (+) plasmid.The Gal 4 sequence, 3×HA sequence were linked into the pcDNA3.1 (+) plasmid with the help of homologous recombination experiments. Special primers were designed and then the plasmid was cut into linear sequences of about 5600bp by PCR.The two ends of this linear sequence can be complementary to the two ends of the KRAB homologous recombination fragment , and the purpose is to conduct homologous recombination with the KRAB homologous recombination fragment to obtain the target product pcDNA3.1 (+) -3 HA-Gal 4-KRAB-NLS plasmid.

1 Pattern Diagram

Fig.1 The agarose gel electrophoresis of pcDNA3.1(+)-3×HA-Gal4-KRAB-NLS linearized vector

2 Experiment

Based on the sequence of the pcDNA3.1 (+) plasmid, With the help of the GAL 4 homologous recombination fragments and homologous recombination experiments.We designed and synthesized the target product.

3.Caution

The two ends of the pcDNA3.1(+)-3×HA-Gal4-VP64-NLS linearized vectror need to be complementary to the two ends of the corresponding recombinant fragments to ensure the success of homologous recombination ligation.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 46
  Illegal XbaI site found at 85
  Illegal SpeI site found at 4849
  Illegal PstI site found at 51
  Illegal PstI site found at 1480
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 46
  Illegal SpeI site found at 4849
  Illegal PstI site found at 51
  Illegal PstI site found at 1480
  Illegal NotI site found at 72
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 46
  Illegal BglII site found at 4612
  Illegal BamHI site found at 6048
  Illegal XhoI site found at 79
  Illegal XhoI site found at 5818
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 46
  Illegal XbaI site found at 85
  Illegal SpeI site found at 4849
  Illegal PstI site found at 51
  Illegal PstI site found at 1480
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 46
  Illegal XbaI site found at 85
  Illegal SpeI site found at 4849
  Illegal PstI site found at 51
  Illegal PstI site found at 1480
  Illegal NgoMIV site found at 590
  Illegal NgoMIV site found at 1931
  Illegal NgoMIV site found at 2216
  Illegal AgeI site found at 143
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5737
  Illegal BsaI.rc site found at 3744
  Illegal BsaI.rc site found at 5482
  Illegal SapI site found at 2661
  Illegal SapI.rc site found at 7
  Illegal SapI.rc site found at 1780
  Illegal SapI.rc site found at 1990