Materials & Method: page1

Primers

The following primers were produced by Sigma-Aldrich (option desalt)

Yeast cloning:

• PKD46-FOR:

5’-  gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggctgccacctgcatcgatttat-3’

• PKD46-REV:

5’-cgtgagttttcgttccactgagcgtcagaccccgtagaaagagttttcgttccactgagc-3’

• PFL-FOR:

5’-Gcctttttacggttcctggc-3’

• PFL-REV:

5’-tttctacgggggtctgacgc-3’

• PCP-FOR:

5’-tggctcttgtatctatcagtgaagcatcaagactaacaaatcagccaaacgtctcttcag-3’

• PCP-REV:

5’-ggggctgtatgcacaaagcatcttctgttgagttaagaacttatatgcgtctatttatgtagg-3’
In green is the 40 homologous nucleotides need for the yeast cloning.

Yeast cloning verification:

• FLP/CI-FOR:

5’-Acatggcgagttttgacgag-3’

• FLP/CI-REV:

5’-accacactagagaacatactg-3’

pINDEL sequencing:

• pID-seq1:

5’-aggatcttcacctagatcctt-3’

• pID-seq2:

 5’-gatgggctagtcaatgataatta-3’

• pID-seq3:

5’-ccgttacgtaggtaggaatc-3’

• pID-seq4:

5’-agatggggatggggcagtc-3’

• pID-seq5:

5’-gatttcggatcaacgttcttaat-3’

• pID-seq6:

5’-caatcactttcgtctactcc-3’

• pID-seq7:

 5’-ccagatatttcgccgcgac-3’

• pID-seq8:

5’-cggggccagcaaaaaatcca-3’

• pID-seq9:

5’-ccctgatttttcaccacccc-3’

• pID-seq10:

5’-cttccgaaaatgcaacgcga-3’

Biobrick frt’-cm-frt’

• frt’-cm-frt’-for: 5’-Gaagttcctatactttttagagaataggaacttcgttgatcgggcacgtaagagg-3’
• frt’-cm-frt’-rev :5’-GAAGTTCCTATTCTCTAAAAAGTATAGGAACTTCTTATTACGCCCCGCCCTGCC-3’

In green is the frt’ sequence which is not homologous with the chloramphenicol gene.

• topo-frt’-cm-frt’-for: tccggcaaaaaagggcaaggtgtcaccaccctgcccttttCgccagtgtgctggatttc
• topo-frt’-cm-frt’-rev: TGCAGCGGCCGCTACTAGTACTCTAGAAGCGGCCGCGAATGATGGATATCTGCAGATTTC

In red is the iGEM restriction sites (prefix and suffix) and in green is the mutation made to suppress the EcoRI restriction site (in the Topo® XL PCR plasmid).

Biobrick frt’-cm-frt’ sequencing (primer M13 as described in Topo® XL PCR Kit)

• FRT’-cm-FRT’-SEQ-FOR:

5’-Gaggaaacagctatga-3’

• FRT’-cm-FRT’-SEQ-REV:

 5’-gaccggcagcaaatg-3’