Plasmid Construction and Gibson cloning

Obtain the sequences of all cDNAs by amplifying existing plasmids, HAP1 cDNA, or through gene synthesis (Genscript).

For insect cell expressions, the sequences are codon optimized and the gene is synthesized (Genscript).

With the exception of the NAP1-6xAla mutant, which was obtained through gene synthesis (Genscript), all other plasmids were generated by Gibson cloning.

For Gibson cloning, generate inserts and vector backbones by PCR amplification or excise from agarose gels after restriction enzyme digestion at 37°C for 2h 0m 0s.

Purify the inserts and plasmid backbones with Promega Wizard SV gel and PCR Cleanup System (Promega).

Mix the purified inserts and backbones in a molar 3:1 ratio, respectively, supplemented by a 2x NEBuilder HiFi DNA assembly enzyme mix (New England Biolabs).

Incubate Gibson reactions for 1h 0m 0s at 50°C and then transform into DH5-alpha competent E. coli cells.

Grow transformed Gibson reactions 1h 0m 0s on agar plates containing the appropriate selection marker (ampicillin, kanamycin, or chloramphenicol).

Pick single colonies, grown 1h 0m 0s in liquid cultures, and pellet for DNA plasmid extraction using the GeneJet Plasmid Miniprep kit (Thermo Fisher).

Submit the purified plasmid DNA for DNA Sanger sequencing (MicroSynth AG) to verify insert sequences by Sanger sequencing.

Further analyze positive clones by whole plasmid sequencing (Plasmidsaurus).