Difference between revisions of "Part:BBa K4585007"
 
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<partinfo>BBa_K4585007 short</partinfo>
 
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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.
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The purpose of the linear vector is to perform homologous recombination with the KRAB homologous recombination fragment to obtain the target product pcDNA3.1(+)-3×HA-GAL4-KRAB-NLS plasmid.
 
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                 pcDNA3.1(+)-3×HA-GAL4-VP64-NLS
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                 pcDNA3.1(+)-3×HA-GAL4-VP64-NLS linearized vector
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                 <p>The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid was obtained through homologous recombination of the VP64 homologous recombination insert (BBa_K4585002) with pcDNA3.1(+)-3×HA-GAL4-VP64-NLS linearized vector (BBa_K4585006).  The homologous recombination plasmid product was identified as the target product by sequencing and enzyme cutting and agarose gel electrophoresis.
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                 <p>We based on the sequence of the pcDNA3.1(+) plasmid.The GAL4 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 5600 bp 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-GAL4-KRAB-NLS plasmid.
 
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                 1 Pattern Diagram
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                 1 Diagrams
 
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                     <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/the-model-diagram-of-pcdna3-1-3-ha-gal4-vp64-nls.png"></p>
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                     <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/pcdna3-1-3-ha-gal4-krab-nls-linearized-vector/pcdna3-1-3-ha-gal4-krab-nls-linearized-vector.png"></p>
 
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                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.1 The model diagram of pcDNA3.1(+)-3×HA-GAL4-VP64-NLS</p>
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                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig 1. The agarose gel electrophoresis of pcDNA3.1(+)-3×HA-GAL4-KRAB-NLS linearized vector</p>
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                 2 Experiment
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                 2 Caution
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                 2.1 Method
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                 <p>The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid could express GAL4-VP64, thereby activating 9×UAS, which could activate the expression of its downstream gene, GAL4-KRAB or Luciferase.
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                 <p>The two ends of the pcDNA3.1(+)-3×HA-GAL4-KRAB-NLS linearized vector need to be complementary to the two ends of the corresponding recombinant fragments to ensure the success of homologous recombination ligation.
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                2.2 Results
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                <p>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.
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                    <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/biofluorescence-intensity-when-gal4-vp64-gal4-krab-400-ng.png"></p>
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                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig 2. Bioluminescence intensity when GAL4-VP64=GAL4-KRAB=400 ng</p>
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                3.Caution
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                <p>After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4585007 SequenceAndFeatures</partinfo>

Latest revision as of 03:45, 12 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(+)-3×HA-GAL4-KRAB-NLS plasmid.

pcDNA3.1(+)-3×HA-GAL4-VP64-NLS linearized vector

We based on the sequence of the pcDNA3.1(+) plasmid.The GAL4 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 5600 bp 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-GAL4-KRAB-NLS plasmid.

1 Diagrams


Fig 1. The agarose gel electrophoresis of pcDNA3.1(+)-3×HA-GAL4-KRAB-NLS linearized vector

2 Caution

The two ends of the pcDNA3.1(+)-3×HA-GAL4-KRAB-NLS linearized vector 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