Coding

Part:BBa_K1499201:Design

Designed by: Raman Nelakanti   Group: iGEM14_StanfordBrownSpelman   (2014-08-25)
Revision as of 22:23, 23 October 2014 by Raman2004 (Talk | contribs)

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Deinococcus radiodurans uracil-DNA glycosylase 1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 720
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part was isolated from the genomic DNA of Deinococcus radiodurans (1). First, genomic DNA was extracted from cells growing in an overnight liquid culture. The gene was simultaneously amplified from genomic DNA and added BioBrick ends using the following protocol: 

Forward primer uracil-DNA glycosylase 1 (46 bp)

5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGATGACCGACCAACCCGAC-3'

Reverse primer uracil-DNA glycosylase 1 (50 bp)

5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTCCTCCGTCACCGTGGC-3'

60 ng genomic DNA/reaction 

1 uM primer mix 

25 ul AmplitaQ gold in a total reaction volume of 50 uL 

1 min of 95C denaturation and 45 sec of annealing, x34 cycles


Note 1: The stop codon was modified from the wild-type TGA to the registry's standard TAA using the reverse amplification primer.

Note 2: This coding part does not contain a promoter, RBS, transcriptional terminator, etc. These elements must be added for proper functionality.

Source

Isolated directly from Deinococcus radiodurans genomic DNA, Chromosome 1 [http://www.ncbi.nlm.nih.gov/genome/1020?genome_assembly_id=170301 NCBI Genome]

References

1. White, O et al. (1999) Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1 Science 286(5444):1571-7. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/10567266 10567266].
2. Sandigursky, M et al. (2004) Multiple uracil-DNA glycosylase activities in Deinococcus radiodurans. DNA Repair (Amst) 3(2):163-9. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/14706350 14706350].