Abstract The retrotransposon Long Interspersed Element 1 (L1) contains adenosine rich ORFs, a characteristic that limits its expression in mammalian cells. A synthetic mouse L1 (smL1) with ORF adenosine content decreased from 40% to 26% showed increased mRNA expression and retrotransposed far more efficiently than the native parental element, L1spa (1). Here, we observe two nonsynonymous substitutions between the L1spa and smL1 ORF1 sequences, and note that the smL1 3’UTR lacks a conserved guanosine-rich region (GRR) which could potentially take on a G-quadruplex secondary structure. We find that the combined effect of a single amino acid change and the GRR 3’UTR deletion, rather than synthetic ORF sequences, accounts for the increase in smL1 retrotransposition efficiency over L1spa. Furthermore, we demonstrate that the position of the GRR within the L1 reporter construct impacts retrotransposition efficiency. Our results prompt a reevaluation of synthetic L1 activity and suggest native mouse L1 mobility has in some cases been underestimated in engineered retrotransposition assays. Author Summary L1 retrotransposons are mobile DNA elements or “jumping genes” that can copy- and-paste their sequences to new locations in the host genome. The jumping ability, or retrotransposition efficiency, of individual L1 elements can be evaluated using a cultured cell assay in which the L1 is tagged in its 3’ untranslated region (3’UTR) with a reporter gene that becomes expressed upon successful retrotransposition. In a previous study, authors Han and Boeke reported that the retrotransposition efficiency of a mouse L1 element could be enhanced dramatically by synthetically increasing the GC content of the L1 ORFs without changing their amino acid sequence. Curiously, a similarly constructed synthetic human L1 achieved only a modest increase in retrotransposition efficiency over the native element. Here, we find that two coding changes and partial deletion of the mouse L1 3’UTR sequence which occurred during construction of the synthetic mouse L1 reporter actually are responsible for the increased jumping of this construct. We also find that changing the placement as well as the presence of this deleted 3’UTR region within the reporter construct determines its impact on engineered retrotransposition efficiency. Together, our study reconciles the disparate impacts of synthetic sequences upon human and mouse L1 retrotransposition efficiency, prompts a reconsideration of numerous studies using synthetic L1 constructs, and will inform the ongoing use of synthetic and natural mouse L1 reporter constructs in vivo and in vitro.