The two 200-mer oligo pools (Twist Bioscience) were first cloned into pLS-mP vector backbone, before sub-cloning into pGL4.23 (Promega) vector backbone. pLS-mP was digested with SbfI and EcoRI (New England Biolabs) to release the minimal promoter-driven eGFP gene. Oligo pools were PCR amplified using primers that are specific to the 5' and 3' flanking sequences and introduce overhangs complementary to the SbfI and EcoRI digested pLS-mP vector backbone. Post gel extraction cleanup (Zymo Research), amplified products were cloned using HiFi DNA Assembly master mix (New England Biolabs). The overhangs complementary to the vector backbone were designed to disrupt the original SbfI and EcoRI sites in the vector post-assembly. The cloning reaction was transformed into electrocompetent Stable cells, and transformants (>5 million/pool)  were pooled, grown overnight and maxi-prepped (Thermo Fisher Scientific) to generate the pre-reporter MPRA plasmid libraries for each of the two oligo pools (Pre-reporter/pLS-mP/Stable). These plasmid libraries were then used to generate the oligo sequencing Illumina libraries and sub-cloning of oligos into pGL4.23 vector backbone. We generated these cloned oligo libraries for potential use in future for lentiviral delivery-based MPRA. For sub-cloning of oligos into pGL4.23, pGL4.23 DNA was first digested with KpnI and XbaI (New England Biolabs) to release the minimal promoter-driven firefly luciferase gene and generate the vector backbone for cloning of MPRA oligos. The cloned oligos in the two pre-reporter MPRA plasmid libraries in pLS-mP backbone were PCR amplified using primers that are specific to the 5' and 3' flanking sequences and introduce overhangs complementary to the KpnI and XbaI digested pGL4.23 vector backbone. Post gel extraction cleanup (Zymo Research), amplified products were cloned using HiFi DNA Assembly master mix (New England Biolabs). The cloning reaction was transformed into electrocompetent Stable and DH10β cells, and transformants (>5 million/pool) were pooled, grown overnight and maxi-prepped (Thermo Fisher Scientific) to generate two versions, V1 and V2, of the pre-reporter MPRA plasmid libraries for each of the two oligo pools (V1: Pre-reporter/pGL4.23/Stable; V2: Pre-reporter/pGL4.23/DH10β). Two different E. coli host cells were used for library propagation to maximize representation and recovery of test elements. These pre-reporter plasmid libraries were digested with SbfI and EcoRI (New England Biolabs), sites for which are present only in the deigned oligos, and ligated with the minimal promoter-driven eGFP reporter cassette from pLS-mP, released by SbfI and EcoRI digestion. Like above, the cloning reaction was transformed into electrocompetent Stable and DH10β cells, and transformants (>5 million/pool) were pooled, grown overnight and maxi-prepped (Thermo Fisher Scientific) to generate two versions, V1 and V2, the post-reporter MPRA plasmid libraries for each of the two oligo pools (V1: Post-reporter/pGL4.23/Stable; V2: Post-reporter/pGL4.23/DH10β). Quality of the synthesized oligos and their representation was assessed by high throughput sequencing. For each of the two oligo pools, oligos cloned in the pre-reporter MPRA plasmid libraries (Pre-reporter/pLS-mP/Stable) were amplified using custom primers that include P5 and P7 Illumina adapter sequences to generate custom amplicon single-indexed oligo sequencing libraries. These two libraries were pooled and sequenced on Illumina MiSeq platform (2×170 + 8 base index) using custom sequencing primers to cover the entire oligo except for the 5' and 3' constant flanking sequences. The post-reporter MPRA plasmid libraries (V1 and V2) were transfected using FuGENE HD Transfection Reagent (Promega) into HL-1 cells, 24h post seeding of 5 million cells in 10 cm dishes, in 10 parallel replicates per pool to ensure robustness and reproducibility. To preserve library complexity, the number of cells at the time of transfection ensures that at least 50× more cells get transfected as compared to the number of unique oligos after adjusting for ~40% transfection efficiency. Forty-eight hours post- transfection cells were harvested, and total RNA was isolated using QIAshredder (Qiagen) and RNeasy Mini Kit (Qiagen). All RNA isolated was used to isolate mRNA using oligo-dT based Magnetic mRNA Isolation Kit (New England Biolabs), which was then treated with TURBO DNase (Thermo Fisher Scientific) to remove contaminating DNA. 500 ng of mRNA from each replicate was reverse transcribed with SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) in two parallel reactions using a primer downstream of the barcode, which contained a sample index and P7 Illumina adapter sequence. The two cDNA preparations for each replicate transfection were combined, and used as a template in PCR to generate the cDNA (RNA) -based MPRA barcode sequencing library with the same reverse primer and a forward primer complementary to the 3' end of eGFP with a P5 Illumina adapter sequence. Similarly, the post-reporter MPRA plasmid libraries (V1 and V2) for each oligo pool were used as templates in PCR to generate the input plasmid (DNA) -based MPRA barcode sequencing libraries in three replicates. In total, we generated 26 (3 replicates for input plasmid DNA and 10 transfection replicates for RNA for each of the two oligo pools) custom amplicon single-indexed barcode libraries for V1, which were pooled and sequenced on the Illumina NextSeq 550 platform (2×26 + 8 base index; high-output kit) using custom sequencing primers. The same was repeated for V2.