coding sequence of mneongreen  (GenScript corporation)

Journal: bioRxiv

Article Title: Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

doi: 10.1101/694190

Figure Lengend Snippet: Absorbance (dashed lines) and emission spectrum (solid lines) of purified mNeonGreen (green lines) overlaid with the spectrum of purified mRuby3 (A) , mScarlet-I (B) , and mCherry (C) . Spectrum were obtained from the reference indicated in .

Article Snippet: NG-Ruby3 was created by removing mClover3 and all but the last 5 amino acids of the linker from pKanCMV-mClover3-mRuby3 by inverse PCR, and the full mNeonGreen coding sequence was inserted in its place using In-Fusion cloning (Takara Bio; Mountain View, CA).

Techniques: Purification

Journal: bioRxiv

Article Title: Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

doi: 10.1101/694190

Figure Lengend Snippet: (A) Cartoon schematic of the mNeonGreen-Red FP tandem constructs used for FRET experiments. (B) Average emission scan of cells expressing NG-Stop (black) when excited at 470 nm overlaid with the reported spectrum for purified mNeonGreen in green (n = 3 independent transfections). Example raw emission spectrum (pink) of tandem (C) NG-Ruby3, (D) NG-Scarlet, and (E) NG-Cherry when excited at 470 nm. The dashed black line shows the sum of donor (green) and acceptor (red) components calculated by linear unmixing. (F) FRET efficiencies calculated from the spectrum for each construct (n=3). *** = P < 0.0005 between the indicated conditions.

Article Snippet: NG-Ruby3 was created by removing mClover3 and all but the last 5 amino acids of the linker from pKanCMV-mClover3-mRuby3 by inverse PCR, and the full mNeonGreen coding sequence was inserted in its place using In-Fusion cloning (Takara Bio; Mountain View, CA).

Techniques: Construct, Expressing, Purification, Transfection

Journal: bioRxiv

Article Title: Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

doi: 10.1101/694190

Figure Lengend Snippet: (A) Lifetime data collected from individual HEK293 cells expressing cytosolic mNeonGreen at various laser powers up to 25 W/cm 2 after 50 frames. Black bars indicate the average ± 95% confidence interval. (B) Lifetime and (C) intensity of samples taken over 300 frames at various laser powers. * = P < 0.05, ** = P< 0.005, and *** = P < 0.0005 compared to the frame matched 5W/cm 2 dataset. N for each sample is as follows 5W/cm 2 : 11 cells, 10W/cm 2 : 10 cells, 15W/cm 2 : 14 cells, 20W/cm 2 : 9 cells, 25W/cm 2 : 11 cells. (D) Example lifetime decay curves obtained at 15 W/cm 2 over 300 frames. (E) Example lifetime decay and normalized decays (F) obtained at 25 W/cm 2 over 300 frames.

Article Snippet: NG-Ruby3 was created by removing mClover3 and all but the last 5 amino acids of the linker from pKanCMV-mClover3-mRuby3 by inverse PCR, and the full mNeonGreen coding sequence was inserted in its place using In-Fusion cloning (Takara Bio; Mountain View, CA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Comparing the Performance of mScarlet-I, mRuby3, and mCherry as FRET Acceptors for mNeonGreen

doi: 10.1101/694190

Figure Lengend Snippet: (A) The lifetime of mNeonGreen in individual cells expressing a mNeonGreen-Red Protein tandem fusion construct. Black bars indicate the average ± 95% confidence interval. (B) FRET efficiency calculations for each tandem construct. *** = P < 0.005 compared to NG-Stop and # indicates P < 0.0005 compared to NG-Stop and P < 0.0005 compared to NG-Ruby3. NG-Scarlet and NG-Cherry are not statistically different (P=0.75) (C) Example decay curves for each tandem representative of the average lifetime of all cells for each construct. (D) Example lifetime heat maps for a single frame for each construct. N for each construct is as follows NG-Stop: 68 cells, NG-Ruby3: 63 cells, NG-Scarlet: 64 cells, and NG-Cherry: 64 cells.

Article Snippet: NG-Ruby3 was created by removing mClover3 and all but the last 5 amino acids of the linker from pKanCMV-mClover3-mRuby3 by inverse PCR, and the full mNeonGreen coding sequence was inserted in its place using In-Fusion cloning (Takara Bio; Mountain View, CA).

Techniques: Expressing, Construct