Part:BBa_K3629026

mCitrine expression construct with Gibson homology

mCitrine expression construct with Gibson homology, TEF intronic promoter (BBa_K3629001)XPR2 terminator(BBa_K3629004), codon optimized mCitrine coding sequence (BBa_K3629022) and a pair of gPCR primer binding site for Yarrowia lipolytica.

Usage and Biology

The construct is designed for optimal expression of mCitrine (BBa_K3629022) in Yarrowia lipolytica.

mCitrine (BBa_K3629022) is a monomeric green fluorescent protein that is derived from Aequorea victoria. mCitrine's optimal excitation wavelength is 515nm and its optimal emission wavelength is 529nm (1). mCitrine could be used to tag components and proteins within the cell which can then be visualized using fluorescence spectroscopy and microscopy.

Expression of mCitrine 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.

mCitrine coding sequence was also codon-optimized for expression and function in Yarrowia lipolytica.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1629
  Illegal SpeI site found at 331
  Illegal PstI site found at 296
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1629
  Illegal NheI site found at 121
  Illegal SpeI site found at 331
  Illegal PstI site found at 296
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1629
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1629
  Illegal SpeI site found at 331
  Illegal PstI site found at 296
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1629
  Illegal SpeI site found at 331
  Illegal PstI site found at 296
  Illegal NgoMIV site found at 1345
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Lambert, T. (n.d.). MCitrine at FPbase. Retrieved October 26, 2020, from https://www.fpbase.org/protein/mcitrine/

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