title: GSM7251911: GN_attP_GG_cells_replicate1; synthetic construct; OTHER
library_construction_protocol: HEK 293T genomic DNA was extracted using a Qiagen DNeasy Blood and Tissue Kit The DNA segments of interest were amplified from 500 ng DNA with 0.3125 μM forward and reverse primer in a 40 μL reaction with Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific, F548L) added as half the volume. Reaction conditions were 95℃ 3', 95℃ 15”, 60℃ 15”, 72℃ 30”, repeated twenty-six to twenty-eight times, 72℃ 1', 4℃ hold. In the case of excision via flanking GA sites, an extra forward or reverse primer was used so that both excised and unexcised products could be amplified from the same PCR tube. PCR products were run on a 2% TAE-agarose gel. DNA bands were excised and extracted using a GeneJet Gel Extraction and DNA Cleanup Micro Kit. The DNA regions of interest were sequenced by the Amplicon-EZ service provided by Genewiz (now Azenta Life Sciences), providing Illumina high throughput sequencing  with read 1 and read 2 sequences of 250 nt each OTHER: Amplicon sequencing of DNA recombined within cells