Correct option is D
The correct answer is (d).
The correct construct for expressing a gene of interest (Goi) and a green fluorescent protein (GFP) independently but from a single mRNA transcript in mammalian cells must have the enhancer (Enh) and promoter (Pro) upstream for proper transcription initiation. The gene of interest (Goi) is placed immediately downstream for translation to start normally. The Internal Ribosome Entry Site (IRES) is a crucial element that allows ribosomes to initiate translation internally on the mRNA, enabling the independent translation of the downstream GFP. The polyadenylation signal sequence (pA) is at the 3' end to stabilize the mRNA. Thus, the order must be: Enh – Pro – Goi – IRES – GFP – pA (Option d).
Incorrect option-
Option (a) is incorrect because placing IRES before GFP is correct, but enhancer and promoter order should be enhancer first then promoter; otherwise, transcription efficiency could be affected.
Option (b) is incorrect because GFP is placed before IRES, so GFP translation would be dependent on the upstream cistron and not independent.
Option (c) is incorrect because GFP is placed before IRES, which will prevent independent translation of GFP.
