Part:BBa_M50042:Design
HIV I Integrase pD649 used in DNA 2.0
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 70
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 205
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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/