Untranslated regions at the 3' end of the messenger RNA (3' UTR) contain regulatory elements that affect mRNA stability and translation and subsequently the protein levels. In this report, we performed bioinformatics analysis on housekeeping genes with putative stable mRNAs in comparison with Class II AU-rich elements (ARE)-containing mRNAs, a group of mRNAs known to represent many labile transcripts. We have found that ARE-mRNAs are less abundant and had longer 3' UTR than stable housekeeping mRNAs. As a result of the analysis, we evaluated the use of a 3' UTR derived from the abundant elongation factor 1 alpha 1 (EEF1A1) mRNA, in expression vectors. Due to the excellent consequence of the modified 3' UTR, we were able to produce expression active linear DNA generated by cloning-free PCR. We have also applied this approach to study the in vivo minimum requirement of poly(A) signal context that allows efficient protein synthesis. The efficient 3' UTR may find use in enhanced recombinant protein production and also provide a simplified tool for generation of expression active linear DNA.