Part:BBa_Y00100

nuclear 5-aminolevulinate synthase gene from S. cerevisiae, recoded for mitochondrial expression

The first step in S. cerevisiae's pathway for heme biosynthesis is catalyzed by the product of the HEM1 gene [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=HEM1]. HEM1 is a nuclear gene (1.647 kb,[http://db.yeastgenome.org/cgi-bin/getSeq?seq=YDR232W&flankl=0&flankr=0&map=a3map]) that encodes the enzyme 5-aminolevulinate ("ALA" or "d-ALA") synthase (549 aa,[http://db.yeastgenome.org/cgi-bin/getSeq?seq=YDR232W&flankl=0&flankr=0&map=p3map]). The enzyme is post-translationally imported into the mitochondria where it is responsible for converting glycine and succinyl-CoA to 5-aminolevulinate ("d-ALA") plus CoA and CO2. Deletion of HEM1 is lethal for the cell but viabilty can be restored by providing the cells with the product of the HEM1p catalyzed reaction, namely d-ALA. In an effort to directly express the HEM1 gene from the mitochondrial genome, the HEM1 gene was recoded by DNA 2.0 for mitochondrial codons, an epitope tag (HA = YPYDVPDYA) was added to the C-terminal portion of the protein just before the stop codon, and flanking sequences (100 bp upstm of ATG and 92 dwstm of stop) were added to facilitate integration into mitochondrial genome.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 694
  Illegal BglII site found at 1169
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]