Composite

Part:BBa_K1861070:Design

Designed by: Vladyslav Vyshnevskyi   Group: iGEM15_FAU_Erlangen   (2015-09-18)

Fusion protein of TALE3 and a SpyTag in an expression cassette


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1364
    Illegal NheI site found at 2445
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 260
    Illegal XhoI site found at 2241
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 707
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

As the TALE's repeats proved difficult to synthesize reliably, we used DNAWorks to remove repeats while keeping the same amino acid sequence.
In order to insert the variable TALE domain (VD) into the constant domain (CD) BBa_K1861201, we had to introduce two restriction sites for Esp3I and a spacer between them in the CD, as well as two Esp3I sites in each half of the VD. Thus, we ligated the 5' Esp3I site of the CD with the complementary 5' site of the VD's first half, its 3' site with the VD's second half's 5' site, and VD's second half's 3' site with the CD's 3' site.
We used the BamHI and SacI restriction sites to clone the TALE into the expression cassette BBa_K1861010. The cassette BBa_K1861010 and the four fragments of the TALE BBa_K1861701 have been ordered from IDT.


Source

The TALE sequence was obtained using the TAL Plasmids Sequence Assembly Tool.
The rpd3 gene is located on chromosome XIV of S. cerevisiae. The promoter sequence can be found in the Yeast Promoter Atlas.
For the SpyTag and the terminator, we used the sequences of BBa_K1159201 and BBa_K392003, respectively.


References

http://helixweb.nih.gov/dnaworks/, visited on 2015-09-21
http://web.archive.org/web/20150921225027/http://helixweb.nih.gov/dnaworks/
http://bao.rice.edu/Research/BioinformaticTools/assembleTALSequences.html, visited on 2015-09-20 http://web.archive.org/web/20150920175258/http://bao.rice.edu/Research/BioinformaticTools/assembleTALSequences.html
https://eu.idtdna.com/site, visited on 2015-09-19
http://ypa.csbb.ntu.edu.tw/do?act=gene_by_kw&query=rpd3, visited on 2015-09-19
http://bao.rice.edu/Research/BioinformaticTools/assembleTALSequences.html, visited on 2015-09-20
http://web.archive.org/web/20150920175258/http://bao.rice.edu/Research/BioinformaticTools/assembleTALSequences.html