Coding

Part:BBa_M50042:Design

Designed by: Michelle Bae   Group: Stanford BIOE44 - S11   (2016-12-08)
Revision as of 23:21, 8 December 2016 by Hbae (Talk | contribs) (→‎References)


HIV I Integrase pD649 used in DNA 2.0


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 70
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 205
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 277


Design Notes

The integrase plasmid, pD649_Integrase, uses the premade mammalian vector pD649 from DNA 2.0. In this plasmid, mammalian expression is driven by the powerful constitutive promoter and enhancer found in cytomegalovirus, or CMV. After the promoter, the integrase gene from HIV-1 was added. The amino acid sequence for integrase was sourced from the HIV Databases. This sequence was translated into DNA and optimized the codons for human expression using IDT’s codon optimizer. Following the integrase gene, an internal ribosome entry site, or IRES, was used in order to express green fluorescent protein, or GFP. This GFP will act as a reporter protein that can confirm both the successful transfection of the cells and the expression of the integrase protein. Finally, there is a poly-adenosine terminator which will act as a stopping point for transcription. Along with these elements, the plasmid also contains a number of other elements necessary for prokaryotic replication and ampicillin selection. Furthermore, the plasmid also contains puromycin resistance genes and a replication origin for stable expression in mammalian cells; however, neither of these elements will be used in the experiment.

Source

The amino acid sequence for integrase was sourced from the HIV Databases: http://www.hiv.lanl.gov/

References

Mammalian Expression Vectors [Internet]. DNA 2.0 [cited 2016 Oct 30]. Available from: https://www.dna20.com/eCommerce/catalog/datasheet/448 HIV Databases [Internet]. Los Alamos: Los Alamos National Security; c2005-2006 [cited 2016 Oct 30]. Available from: http://www.hiv.lanl.gov/ Codon Optimization Tool [Internet]. Redwood City: Integrated DNA Technologies [cited 2016 Oct 30]. Available from: https://www.idtdna.com/CodonOpt