Part:BBa_K3629025
mCherry expression construct with Gibson homology
mCherry expression construct with Gibson homology, TEF intronic promoter (BBa_K3629001)XPR2 terminator(BBa_K3629004), codon optimized mCherry coding sequence (BBa_K3629021) and a pair of gPCR primer binding site for Yarrowia lipolytica.
Usage and Biology
The construct is designed for optimal expression of mCherry (BBa_K3629021) in Yarrowia lipolytica.
mCherry (BBa_K3629021)is a monomeric red fluorescent protein that is derived from DsRed of Discosoma. mCherry's optimal excitation wavelength is 586nm and its optimal emission wavelength is 610nm (1). mCherry could be used to tag components and proteins within the cell which can then be visualized using fluorescence spectroscopy and microscopy.
Expression of mCherry is under the TEF intronic promoter (BBa_K3629001) which is the natural promoter found in the wild-type Yarrowia lipolytica for the Translation Elongation Factor 1 (TEF1) gene; it is a strong constitutive promoter. It also contains the first intron of the gene for stronger expression (2).
Design
The construct is flanked with two Gibson homology sequences. Upon digestion with BbsI, the construct can be connected to either the LEU4 overproducing construct (BBa_K3629023) or the TRP2 overproducing construct (BBa_K3629024)on the 5' side and to the nourseothricin resistance expression construct (BBa_K3629015) on the 3' side. The Gibson homology sequences can also be used to easily add this fluorescent protein to any construct that contains the same homology sequences.
The construct was designed to be ultimately integrated into the genome of Yarrowia lipolytica. Therefore, a pair of gPCR primer binding sites flank the construct and can be used to help determine the location of the integration of the construct in the genome.
mCherry coding sequence was also codon-optimized for expression and function in Yarrowia lipolytica.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1620
Illegal SpeI site found at 331
Illegal PstI site found at 296
Illegal PstI site found at 1015 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1620
Illegal NheI site found at 121
Illegal SpeI site found at 331
Illegal PstI site found at 296
Illegal PstI site found at 1015 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1620
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1620
Illegal SpeI site found at 331
Illegal PstI site found at 296
Illegal PstI site found at 1015 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1620
Illegal SpeI site found at 331
Illegal PstI site found at 296
Illegal PstI site found at 1015 - 1000COMPATIBLE WITH RFC[1000]
References
1. Lambert, T. (n.d.). MCherry at FPbase. Retrieved October 26, 2020, from https://www.fpbase.org/protein/mcherry/
2.Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Applied and environmental microbiology, 77(22), 7905–7914. https://doi.org/10.1128/AEM.05763-11
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