Composite

Part:BBa_K1608003:Design

Designed by: Ching-Yuan Chen, Yi-Ru Liu, Pei-Hong Chen   Group: iGEM15_Mingdao   (2015-09-10)

SRPK-His/pSB1C3


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 97
    Illegal PstI site found at 926
    Illegal PstI site found at 977
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 926
    Illegal PstI site found at 977
    Illegal NotI site found at 2221
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 189
    Illegal BglII site found at 450
    Illegal XhoI site found at 1910
    Illegal XhoI site found at 2230
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 97
    Illegal PstI site found at 926
    Illegal PstI site found at 977
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 97
    Illegal PstI site found at 926
    Illegal PstI site found at 977
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2084


Design Notes

  • One XbaI site and two PstI sites on the PCR-amplified fragment. To be used as a standard BioBrick part, the sites need to be mutated. Or you can just use EcoRI and SpeI to move this part.


We designed two primers to amplify Pt7-SRPK1-His DNA fragment by PCR from SRPK/pET-29b, followed by EcoRI and SpeI digestion and ligated to pSB1C3. The plasmid DNA has been checked by colony PCR, restriction enzyme check and sequencing confirmation. However, there’re two PstI sites one XbaI site on the amplified sequence. We’re trying to mutate them to fit the standard biobrick parts. The SRPK1 gene is fused with 6XHis tag and driven by Pt7 promoter along with a lac operator downstream.


Primers:

  • 1.Pt7(pET)-EcoRI-XbaI-F: 5'- AAAAAAGAATTCGCGGCCGCTTCTAGAGCACGATGCGTCCGGCGTA -3'
  • 2.His (pET)-SpeI-R: 5'- AGGGAAACTAGTATCAGTGGTGGTGGTGGTGGT -3'


PCR product size: ~2294 bp


Plasmid map:

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For more DNA gel data & sequencing data, please go to our [http://2015.igem.org/Team:Mingdao/Parts page]

Source

The fragment was cloned from SRPK/pET-29b.

References

A human importin-beta family protein, transportin-SR2, interacts with the phosphorylated RS domain of SR proteins. [http://www.ncbi.nlm.nih.gov/pubmed/10713112 J Biol Chem. 2000]