Difference between revisions of "Part:BBa K1431301:Design"
(→‎Design Notes)
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<center>https://static.igem.org/mediawiki/parts/archive/a/ab/20141018014544%21Our_RFC.png</center>
 
<center>https://static.igem.org/mediawiki/parts/archive/a/ab/20141018014544%21Our_RFC.png</center>
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<center>Fig.1  The Machanism of Our Standard</center>
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<center>'''Fig.1  The Machanism of Our Standard'''</center>
  
 
If you want to insert a coding sequence between promoter and polyA, the prefix of forward primer you design must contain '''GAAGACNN<span style="color: red">taaa<span style="color: black">NNNN''' and reverse must contain '''GAAGACNN<span style="color: red">agtt<span style="color: black">NNNN'''. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into promoter and polyA seamless. The PCR product must be such sequence, including the protect bases, shows below.
 
If you want to insert a coding sequence between promoter and polyA, the prefix of forward primer you design must contain '''GAAGACNN<span style="color: red">taaa<span style="color: black">NNNN''' and reverse must contain '''GAAGACNN<span style="color: red">agtt<span style="color: black">NNNN'''. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into promoter and polyA seamless. The PCR product must be such sequence, including the protect bases, shows below.

Revision as of 02:01, 18 October 2014

TRE-3G promoter+SV40 PolyA, an ideal controller of mammalian gene expression with Tet-On 3G protein


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

For BbsI site, we have a series same design sequence. See the mechanism of BbsI site work below.

20141018014544%21Our_RFC.png
Fig.1 The Machanism of Our Standard

If you want to insert a coding sequence between promoter and polyA, the prefix of forward primer you design must contain GAAGACNNtaaaNNNN and reverse must contain GAAGACNNagttNNNN. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into promoter and polyA seamless. The PCR product must be such sequence, including the protect bases, shows below.

PCR_product_by_new_RFC.png
Fig.2 PCR Product Sequence

And the reason why we designed that because we want to make a seamless gather between NLS and protein or any triple nucleotides. That depend on which will make the highest efficiency.

Source

The plasmid was from our instructor, Huangwei's lab. And we design primers to copy down by PCR.

===References===