96 New PCT-
Patent Application based on EP 24 186 635.9 Insempra GmbH Vossius Ref.: AG4141 PCT S3 Abstract The present invention relates to a linear double-stranded deoxyribonucleic acid (dsDNA) molecule comprising one or more Tus
protein (Tus)
binding site(s) at the 5'-terminus, one or more Lac
repressor protein (LacI)
binding site(s) at the 3'-terminus, and a segment comprising
a DNA sequence of interest (DOI) between said Tus
binding site(s) and said LacI binding site(s). The present invention further relates to a non-naturally or naturally occurring
RNA molecule as encoded by or transcribable / transcribed from the linear dsDNA molecule of the invention, and to an expression
DNA cassette comprising a
promoter, an RBS, a GOI encoding a POI, and one or more LacI binding site(s), wherein said
expression cassette does not comprise a terminator sequence between the 3´ end of said GOI and said one or more LacI binding site(s). The present invention also relates to a non-naturally or naturally occurring
RNA molecule as encoded by or transcribable / transcribed from said
expression cassette. The present invention further relates to method of protecting a linear deoxyribonucleic acid (
DNA) molecule from
exonuclease degradation by adding one or more Tus binding site(s) at the first terminus of the
DNA molecule and adding one or more LacI binding site(s) at the other terminus of the DNA molecule. The present invention further relates to a method of synthesizing a
protein of interest (POI) in a
cell-
free protein synthesis (CFPS) reaction mixture by using the (ds)DNA and / or
RNA molecules of the invention, and also to a
cell-free
biological system or CFPS reaction mixture comprising the (ds)DNA,
expression cassette and / or RNA molecules of the invention.