Composite

Part:BBa_K1431301:Design

Designed by: Rifei Chen, Yushan Zhang   Group: iGEM14_SUSTC-Shenzhen   (2014-10-10)
Revision as of 01:58, 18 October 2014 by ChenRifei (Talk | contribs)

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

If you want to insert a coding sequence between NLS, 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 two NLS 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===