Part:BBa_K143005:Design

5' Integration sequence for the epsE locus of B. subtilis

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

The epsE integration sequences were based upon the epsE (aka yveO) gene sequence#1 and the sequence of the upstream region on the chromosome (found using NCBI's sequence viewer). The upstream and epsE gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined. The integration sequences were then produced by PCR cloning with Pfu DNA polymerase

Template

B. subtilis Chromosome

• We thank Dr. Angelika Grundling, Centre for Molecular Microbiology and Infection, Imperial College for providing the chromosome preparation for our PCR

Forward Primer

5'- GCT CTA GAG GGT TTT CTG ATC AAA ATC GG - 3'

Reverse Primer

5'- GGA CTA GTA GTT ACG AAA GGT CAT CTC C -3'

Source

The 5’ integration sequence was taken from the B.subtilis chromosome and is homologous to the chromosme from a few hundred bp upstream of the gene's start codon until 300 bp into the gene. It was produced by PCR cloning with Pfu DNA polymerase

References

<biblio>

1. 1 pmid=9384377

</biblio>