Part 4: DNA Synthesis order, building my first plasmid

1. Finding an appropriate plasmid backbone:

Reading the Yang et al. (2023) article, I saw in their “Materials and Methods” section, that they transfected pLVX-EF1alpha 2xGFP:NES-IRES-2xRFP:NLS to generate NCC-stable cells, so I thought it would be a good backbone for the assembly. This “shuttle vector” was originally described by Mertens et al. (2015) (AddGene ID: 71396) Directly Reprogrammed Human Neurons Retain Aging-Associated Transcriptomic Signatures and Reveal Age-Related Nucleocytoplasmic Defects. Mertens J, Paquola AC, Ku M, Hatch E, Bohnke L, Ladjevardi S, McGrath S, Campbell B, Lee H, Herdy JR, Goncalves JT, Toda T, Kim Y, Winkler J, Yao J, Hetzer MW, Gage FH. Cell Stem Cell. 2015 Oct 6. pii: S1934-5909(15)00408-7. doi: 10.1016/j.stem.2015.09.001. 10.1016/j.stem.2015.09.001 PubMed 26456686

2. Assembly

I selected restriction enzymes BamHI and EcoRI to cut the GFP reporter out, and then I built my DNA insert sequence (POU5F1 gene for Oct4) with sticky ends ideal to connect with the backbone. I I incorporated a Kozak consensus sequence (GCCACC) upstream of the ATG instead of an RBS And to add the 7xHis at the C-terminus of the protein, before the stop codon The design leverages the backbone’s endogenous promoter and polyadenylation signal, so I didn’t incorporate those to the insert

And this is the result

https://benchling.com/s/seq-dcl08hA5mckl7k6SyMfX?m=slm-6JoFb5a3gBdbPekfEuDq

Twist order

I tried ordering, but couldn’t so I made another insert, with a Trp-less GFP like in recitation, and got my Hight Copy cassette from Twist: https://benchling.com/s/seq-tiZifTIvfRfmji3ky9IP?m=slm-Xdd23sZE45BAObyU1yP8