Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Natalie Kuldell
Date created: 2006-08-03 11:00:00
Date modified: 2015-05-08 01:14:54
nuclear 5-aminolevulinate synthase gene from S. cerevisiae, recoded for mitochondrial expression
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_Y00100_sequence (Version 1) |
Description
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.
Notes
codon optimized for mitochondrial gene expression, HA-epitope C term (not N-term where mt import sequences rest), flank coding seq with homology to COX3 gene of mitochondria to allow for integration and disruption of this gene (should make respiration+ cells become respiration-), and finally added BB prefix and suffix to allow cloning into std Registry vectorsSource
direct synthesis from DNA 2.0, summer 2006| Sequence Annotation | Location | Component / Role(s) |
| for recombination to COX3 HA TGA stop codon for recombination to COX3 start codon coding sequence | 1,101 1745,1771 1772,1774 1775,1865 101,103 101,1774 | feature/dna sequence_feature feature/tag tag feature/stop stop_codon feature/dna sequence_feature start_codon feature/start CDS feature/cds |