Part:BBa_K1471010:Design
pCMV - YFP.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 576
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
pCMV
The pCMV-Script vector is derived from a high-copy-number pUC-based plasmid and is designed to allow protein expression in mammalian systems. Mammalian expression is driven by the human cytomegalovirus (CMV) immediate early promoter to promote constitutive expression of cloned inserts in a wide variety of cell lines. Selection is made possible in bacteria by the kanamycin-resistance gene under control of the prokaryotic ß-lactamase promoter. The neomycin-resistance gene is driven by the SV40 early promoter, which provides stable selection with G418 in mammalian cells.
The pCMV-Script vector does not contain an ATG initiation codon. A translation initiation sequence must be incorporated if the DNA fragment to be cloned does not have an initiating ATG codon or an optimal sequence for initiating translation, such as the Kozak sequence [GCC(A/G)CCATGG].
The multiple cloning site (MCS) contains fifteen unique restriction enzyme recognition sites organized with alternating 5´ and 3´ overhangs to allow serial exonuclease III/mung bean nuclease deletions. T3 and T7 RNA polymerase promoters flank the polylinker for in vitro RNA synthesis. The choice of promoter used to initiate transcription determines which strand of the DNA insert will be transcribed.
The pCMV-Script vector can be rescued as single-stranded (ss) DNA. The plasmid contains a 454-nucleotide filamentous f1 phage intergenic region (M13-related) that includes the 307 bp origin of replication. The orientation of the f1 origin in pCMV-Script allows the rescue of antisense ssDNA by a helper phage. This ssDNA can be used for dideoxynucleotide sequencing (Sanger method) or site-directed mutagenesis.
YFP
YFP is a genetic mutant of green fluorescent protein (GFP) originally derived from the jellyfish Aequorea victoria. Its excitation peak is 514 nm and its emission peak is 527 nm. Like the parent GFP, YFP is a useful tool in cell and molecular biology thanks to its properties useful for fluorescence microscopy.
Three improved versions of YFP are Citrine, Venus, and Ypet. They have reduced chloride sensitivity, faster maturation, and increased brightness (defined as the product of the extinction coefficient and quantum yield). Typically, YFP serves as the acceptor for genetically-encoded FRET sensors of which the most likely donor FP is monomeric cyan fluorescent protein (mCFP). The red-shift relative to GFP is caused by a Pi-Pi stacking interaction as a result of the T203Y substitution introduced by mutation, which essentially increases the polarizability of the local chromophore environment as well as providing additional electron density into the chromophore.
For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl− are desired. Here we describe the development of an improved version of YFP named “Venus”. Venus contains a novel mutation, F46L, which at 37°C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl−. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.
Design Notes
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Source
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References
PCMV-Script System - Details & Specifications. (n.d.). Retrieved October 30, 2014.
Yellow fluorescent protein. (2014, October 24). Retrieved October 30, 2014.
A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. (n.d.). Retrieved October 30, 2014.