Difference between revisions of "Part:BBa K4585007"
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
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Revision as of 15:30, 8 October 2023


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