Part:BBa_K4585008

pGL4.35-3xHA-9xGAL4UAS-KRAB-NLS linearized vector

The objective was to synthesize the pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid by homologous recombination with the KRAB homologous recombination fragment.

pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector

We based on the sequence of the pGL4.35 plasmid. Through homologous recombination experiments, the GAL4 sequence, 3×HA sequence were ligated to the pGL4.35 plasmid, and nine UAS sequences were inserted simultaneously. Special primers were designed, and then the plasmid was cut into linear sequences of a length of about 4500 bp by PCR. The two ends of the linear sequence can be complementary to both ends of the KRAB homologous recombination fragment, and the purpose is to perform homologous recombination with the KRAB homologous recombination fragment to obtain the target product pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid.

1 Diagrams

Fig 1. The agarose gel electrophoresis of pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector

2 Caution

The two ends of the pGL4.35-3×HA-9×GAL4UAS-KRAB-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 4423
  Illegal XbaI site found at 151
  Illegal XbaI site found at 1933
  Illegal XbaI site found at 4260
  Illegal XbaI site found at 4495
  Illegal SpeI site found at 4019
  Illegal PstI site found at 3108
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4423
  Illegal NheI site found at 4451
  Illegal SpeI site found at 4019
  Illegal PstI site found at 3108
  Illegal NotI site found at 3087
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4423
  Illegal BglII site found at 4498
  Illegal BamHI site found at 413
  Illegal XhoI site found at 4303
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4423
  Illegal XbaI site found at 151
  Illegal XbaI site found at 1933
  Illegal XbaI site found at 4260
  Illegal XbaI site found at 4495
  Illegal SpeI site found at 4019
  Illegal PstI site found at 3108
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4423
  Illegal XbaI site found at 151
  Illegal XbaI site found at 1933
  Illegal XbaI site found at 4260
  Illegal XbaI site found at 4495
  Illegal SpeI site found at 4019
  Illegal PstI site found at 3108
  Illegal NgoMIV site found at 55
  Illegal NgoMIV site found at 166
  Illegal NgoMIV site found at 1644
  Illegal NgoMIV site found at 1661
  Illegal NgoMIV site found at 1793
  Illegal NgoMIV site found at 1884
  Illegal NgoMIV site found at 2136
  Illegal AgeI site found at 1939
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 434
  Illegal SapI site found at 2145