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Part:BBa_M31903:Design
902 with gene 10 promoter
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1299
Illegal EcoRI site found at 1636
Illegal EcoRI site found at 2321
Illegal PstI site found at 1785
Illegal PstI site found at 1917 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1299
Illegal EcoRI site found at 1636
Illegal EcoRI site found at 2321
Illegal PstI site found at 1785
Illegal PstI site found at 1917 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1299
Illegal EcoRI site found at 1636
Illegal EcoRI site found at 2321
Illegal BamHI site found at 3042 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1299
Illegal EcoRI site found at 1636
Illegal EcoRI site found at 2321
Illegal PstI site found at 1785
Illegal PstI site found at 1917 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1299
Illegal EcoRI site found at 1636
Illegal EcoRI site found at 2321
Illegal PstI site found at 1785
Illegal PstI site found at 1917 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
The M13 is extremely compact, which has its adaptational advantages; however, this conciseness presents engineering difficulties when it comes to refactoring. Many genes overlap with each other. Some share stop codons and some contain the ribosome binding sites and promoters of other genes. The refactoring experiment we have undertaken tries to separate these genes such that they can be studied more individually and precisely. The method by which this is done is known as “unstuffing.” For gene 2 and 10: Genes 2 and 10 share a stop codon. Thus, the ORF, RBS, and promoter for gene 10 are all wholly contained within gene 2. A duplicate of the ORF, RBS, promoter sequence for gene 10 was inserted after the stop codon of gene 2. In addition, the ORF, RBS, and promoter sequences that are still embedded within gene 2 were altered such that the amino acds remained the same but the base code was different. This prevents homologous recombination and the subsequent deletion of DNA. Moreoever, restriction sites were added between gene 2 and the new copy of gene 10. The enzymes were added so that one can easily isolate genes when needed. In addition, two restriction sites were added between each gene. Two identical sites flank each gene, so that when a gene is deleted the plasmid can reseal. This process of unstuffing, base changing, and restriction site addition was repeated for all genes in the region of the M13 genome between the unique BamHI site and the unique HapI site.
Source
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