Journal: bioRxiv

Article Title: Kozak sequence libraries for characterizing transgenes across expression levels

doi: 10.1101/2025.04.28.651141

Figure Lengend Snippet: A) Schematic of the HEK 293T landing pad system, with a single plasmid molecule (bottom) integrating into a single genomically engineered site (top). B) Schematic of the transgenic DNA present following plasmid integration. A degenerate Kozak sequence library precedes ACE2 and miRFP670. A histone-fused mCherry and puromycin resistance gene (pac) is encoded behind an IRES and are translated independent of the Kozak sequence. Ptet, Tet inducible promoter; IRES, internal ribosome entry site; upside down red triangle, 2A translation stop-start sequence; brown and black stars, Bxb1 attP and attB recombination sequences, respectively. C) Representative blue and red fluorescence flow cytometry profiles for unmodified HEK 293T cells (black), unrecombined landing pad cells (blue), and antibiotic selected recombined cells (red). D) Representative near infrared fluorescence from high miRFP670 expressing cells treated with a titration of doxycycline inducer (left), or cells expressing miRFP behind Kozak sequences of various translational strengths (right). E) Cell surface ACE2 protein staining of cells expressing the ACE2-Kozak library (green) or control conditions lacking the primary antibody (purple), and unmodified HEK 293T cells mixed with both primary and secondary antibodies (brown).

Article Snippet: Additionally, the following plasmids were deposited in Addgene as part of this work: G790A_AttB_ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR (Addgene plasmid # 237440) and G868A_AttB_[mutATG]ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR (Addgene plasmid # 237441).

Techniques: Plasmid Preparation, Transgenic Assay, Sequencing, Fluorescence, Flow Cytometry, Expressing, Titration, Staining, Control

Journal: bioRxiv

Article Title: Kozak sequence libraries for characterizing transgenes across expression levels

doi: 10.1101/2025.04.28.651141

Figure Lengend Snippet: A) Cells encoding the ACE2-Kozak library (green) were compared to cells encoding ACE2 behind a weak (blue) or strong (red) translation Kozak sequences, and assessed for near infrared miRFP670 fluorescence using flow cytometry. The ACE2-Kozak library cell population was broken into quartile bins using threshold values (dotted lines), and the distribution of the control cells amongst the bins are shown as percentages. B) Schematic of the Sort-seq approach used to calculate steady-state abundance values corresponding to each Kozak variant. C) The distribution of DNA sequencing reads corresponding to the control Kozak sequences amongst the four bins for 5 replicate experiments, as well as the calculated mean weighted average corresponding to each sequence. D) Histogram of the mean weighted averages for all Kozak variants in the dataset.

Article Snippet: Additionally, the following plasmids were deposited in Addgene as part of this work: G790A_AttB_ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR (Addgene plasmid # 237440) and G868A_AttB_[mutATG]ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR (Addgene plasmid # 237441).

Techniques: Fluorescence, Flow Cytometry, Control, Variant Assay, DNA Sequencing, Sequencing

Journal: bioRxiv

Article Title: Kozak sequence libraries for characterizing transgenes across expression levels

doi: 10.1101/2025.04.28.651141

Figure Lengend Snippet: A) Comparison of our calibrated abundance scores with mean Kozak translational scores from the Noderer et al. dataset. B) Geometric means of miRFP670 mean fluorescence intensity measured when the indicated Kozak sequences were tested individually, and comparison with the Noderer et al. dataset. C) Comparison of the random-forest-imputed abundance scores with the Noderer et al translational efficiency scores. Two subtypes of Kozak sequences encoding upstream open reading frames starting at the −5 position, both defying the correlative pattern of the overall dataset, are colored orange and blue. D) Schematic showing the nucleotide sequences upstream of the degenerate Kozak library site, demonstrating the sequence features likely underlying reasons for discordance between the two datasets. uORF#1A and uORF#1B corresponds to the orange and blue points in panel C, respectively.

Article Snippet: Additionally, the following plasmids were deposited in Addgene as part of this work: G790A_AttB_ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR (Addgene plasmid # 237440) and G868A_AttB_[mutATG]ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR (Addgene plasmid # 237441).

Techniques: Comparison, Fluorescence, Sequencing

Journal: bioRxiv

Article Title: Kozak sequence libraries for characterizing transgenes across expression levels

doi: 10.1101/2025.04.28.651141

Figure Lengend Snippet: A) Work-flow diagram describing key physical and computational steps for the multiplex infection assay readout. B) Scatterplots of pseudotyped virus infectivity as a function of calibrated abundance scores. The semi-translucent red lines denote a LOESS fit to the points. The plot to the rightmost column overlay these LOESS curves. The horizontal purple line denotes the infectivity of template plasmid, which encodes a null ACE2 sequence lacking its start codon. C) Scatterplots of pseudotyped virus infectivity as a function of miRFP670 fluorescence of a set of clonally tested Kozak sequences. D) Scatterplot overlays of the same set of Kozak sequences shown in panels 6B and 6C, with normalized infection score as a function of protein abundance. The LOESS fit colors denote the two different assay types compared.

Article Snippet: Additionally, the following plasmids were deposited in Addgene as part of this work: G790A_AttB_ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR (Addgene plasmid # 237440) and G868A_AttB_[mutATG]ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR (Addgene plasmid # 237441).

Techniques: Multiplex Assay, Infection, Virus, Plasmid Preparation, Sequencing, Fluorescence, Quantitative Proteomics

Journal: bioRxiv

Article Title: Kozak sequence libraries for characterizing transgenes across expression levels

doi: 10.1101/2025.04.28.651141

Figure Lengend Snippet: A) Histograms of calibrated scores when the −6 Kozak nucleotide is left invariant. B) Schematic demonstrating the ACE2 sequence variants, their nucleotide identifiers, and their anticipated infection phenotypes (top), as well as a visual depiction of 100-fold receptor density differences expected on a two-dimensional plane, mimicking cell surface density (bottom). C) Scatterplots of pseudotyped virus infectivity as a function of calibrated abundance scores of ACE2 variants. The semi-translucent red, green, and blue lines denote LOESS fits to the points. The scatterplots in the bottom row overlay these LOESS curves. The horizontal purple line denotes the infectivity of template plasmid, which encodes a null ACE2 sequence lacking its start codon. D) Schematic demonstrating the STIM1 sequence variants, their nucleotide identifiers, and their anticipated cell toxicity phenotypes (top), as well as a key denoting how STIM1 abundance level is expected to correspond to cell toxicity (bottom). E) Histograms showing the distribution of cell survival scores, separated by STIM1 variant. F) Overlaid LOESS curves fitting data-points denoting cell survival as a function of STIM1 calibrated abundance score.

Article Snippet: Additionally, the following plasmids were deposited in Addgene as part of this work: G790A_AttB_ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR (Addgene plasmid # 237440) and G868A_AttB_[mutATG]ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR (Addgene plasmid # 237441).

Techniques: Sequencing, Infection, Virus, Plasmid Preparation, Variant Assay