This in vitro transcribed NLS-Cre mRNA encodes a human/mouse codon-optimized Cre recombinase fused to a nuclear localization sequence (NLS). The mRNA is designed for efficient ex vivo loxP-mediated recombination in cell lines and primary cells. Incorporation of Cy5-labeled nucleotides enables direct visualization of mRNA delivery and expression via fluorescence microscopy using a Cy5 filter.

• 5' Cap Structure: ARCA (Anti-Reverse Cap Analog; m⁷(3'-O-methyl)-G(5')ppp(5')G, Cap 0) or CleanCap AG (m⁷G(5')ppp(5')(2'-O-methyl-A)pG, Cap 1) for enhanced mRNA stability and translation.
• Nucleotide Modifications: Cy5-UTP and N¹-methyl-pseudouridine-5'-triphosphate at a 1:3 molar ratio, plus 5-methylcytidine triphosphate (5mCTP), to improve mRNA stability and reduce immunogenicity.
• Poly(A) Tail: 180 nucleotides for optimal translation efficiency.

• Ex vivo loxP site-specific recombination
• Visualization of mRNA delivery and expression by Cy5 fluorescence

• Agarose gel electrophoresis
• Functional assay: HEK293 cells transfected with loxP reporter plasmid