Part:BBa_K1861030:Design

eYFP gene for expression in yeast

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 276
  Illegal XhoI site found at 300
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 967
  

Design Notes

To find usable TALE binding sites, we had to determine which sequences were rare, unique, or in the best case, not present at all in the yeast genome. Using the software we wrote for this task (see http://2015.igem.org/Team:FAU_Erlangen/Software for more information), we scanned the genome of S. cerevisiae s288C for all possible 10 nucleotide long sequences. We succeeded in finding one unique and two nonexistent sequences and usem them to design the following binding sites:

TALE1: T CTCCACCCCATT, appears in the rad14 gene on chromosome 13

TALE2: T GTCGTAACAACT, appears nowhere

TALE3: T CCGACTCGCTGT, appears nowhere.
In case these sequences contradict the TALE binding sequences in our wiki, consider the information given here correct.

In order to clone eYFP into this part, we had to add the restriction sites for XhoI and SalI to BBa_E3020. We used the following primers to achieve this:

5' XhoI primer - TCT GCT CGA GGC AACTAG CGG CAT G

3' SalI primer - CTA CGT CGA CTT ATG CGGTAC CAG AAC CTT TG
The restriction sites are in italic.
As the start codon ATG of BBa_K1861302 lies before the XhoI site, the resulting protein will begin with the amino acids MLE. The start codon ATG of BBa_E2030 was removed by the XhoI primer, as the last G of the restriction site corresponds to the G of the start codon of BBa_E2030.
All sequences apart from BBa_E2030 were ordered from IDT.

Source

The promoter sequence can be found on SnapGene.
The sequence of the nucleotides close to the his3 gene was obtained through the Saccharomyces Genome Database.
For the Kozak sequence and the terminator, we used the sequences of BBa_K792001 and BBa_J63002, respectively.

References

https://eu.idtdna.com/site, visited on 2015-09-19
http://www.snapgene.com/resources/plasmid_files/basic_cloning_vectors/CMV_promoter/, visited on 2015-09-20
http://web.archive.org/web/20150920115303/http://www.snapgene.com/resources/plasmid_files/basic_cloning_vectors/CMV_promoter/ http://www.yeastgenome.org/locus/S000005728/overview, visited on 2015-09-20

http://web.archive.org/web/20150920202914/http://www.yeastgenome.org/locus/S000005728/overview