N50-C random 3′ UTR growth-selection MPRA

A low-copy centromeric episomal reporter library of random 50-bp synthetic sequences was inserted into the 3′ UTR of a HIS3 reporter in the CYC1 promoter/terminator framework. The N50-C design replaced the first 151 bp of the CYC1 3′ UTR while retaining the cleavage site and downstream constant sequence; library-member abundance before versus after 3-AT growth selection provided the expression proxy.

Processed tables are specific to each experiment. Column names, units, measurements, and table structure are not standardized across the database. Check this experiment’s column definitions and quality-control notes before comparing or combining data.

Organism: Budding yeast
Taxonomy ID: NCBITaxon:4932
Biosample: UNMAPPED:BY4741_his3_KanMX
Reference genome: Not reported / not applicable
Design focus: Synthetic / Motif-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

1 mM 3-amino-1,2,4-triazole (3-AT) in SD-Leu-His growth selection; SD-Leu culture was the pre-selection input

The p415-CYC1-HIS3 low-copy centromeric plasmid carried a LEU2 marker and a random 50-bp N50 sequence in the reporter 3′ UTR. N50-C removed the first 151 bp of the native CYC1 terminator and retained its cleavage site plus downstream constant sequence. BY4741 his3::KanMX yeast were grown before selection and to OD660 approximately 1 in SD-Leu-His plus 1 mM 3-AT; pre- and post-selection plasmid amplicons were sequenced on an Illumina NextSeq 550, clustered with Bartender, and aligned with Bowtie2. The read-frequency change is a continuous proxy for HIS3 protein expression and fitness, not a direct RNA/DNA stability measurement.

Quality control

The authors required at least 5 reads in the input sample and at least 1 read in the output sample, with no pseudocounting; this produced 590,024 N50-C variants in the source table. Independent package QC verified 50-nt sequences, an A/C/G/T-only alphabet, unique sequence identities, positive finite counts/frequencies, finite log2 enrichment, and consistency of the source enrichment with log2(output_frequency/input_frequency) within 0.02. All 590,024 source rows passed and were retained.

Curation notes

This library was measured in one pooled growth-selection assay without biological replicates because a replicate transformation would generate a different random library. The sequence itself is the variant identity; there is no separate barcode or allele contrast. N50-C lacks invariant efficiency and positioning elements, so its score distribution is not directly interchangeable with N50-EPC without accounting for library context. Motif flags are derived annotations using DNA T→RNA U conversion and the N/W wildcard conventions stated in the paper. The processed table contains 590024 rows, including the authors’ low- and high-enrichment variants; the raw sequencing reads remain in BioProject PRJNA750726 and are not packaged.

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N50-C random 3′ UTR growth-selection MPRA

Processed data

Quality control

Curation notes

Cite OpenMPRA

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