Nt in dihydrofolate reductase activity. Proc Natl Acad Sci U S A 1980, 77(7):4216?220. 11. Derouazi M, Martinet D, Besuchet Schmutz N, Flaction R, Wicht M, Bertschinger M, Hacker DL, Beckmann JS, Wurm FM: Genetic characterization of CHO production host DG44 and derivative recombinant cell lines. Biochem Biophys Res Commun 2006, 340(four):1069?077. 12. Weiner M, Gackstetter T, Costa G, Bauer C, Kretz K: Site-directed Mutagenesis using PCR. In Molecular Biology: Existing Innovations and Future Trends. Edited by Griffin A, Griffin H. Wymondham, Norfolk, U.K: Horizon Scientific Press; 1995. 13. Orlova NA, Orlov AV, Vorobiev II: A modular assembly cloning approach (aided by the BIOF software tool) for seamless and error-free assembly of long DNA fragments. BMC Res Notes 2012, five:303. 14. Kozak M: An analysis of 5-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 1987, 15(20):8125?148. 15. Patterson GH, Knobel SM, Sharif WD, Kain SR, Piston DW: Use from the green RORγ Inhibitor medchemexpress fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 1997, 73(five):2782?790. 16. Ribeiro S, Mairhofer J, Madeira C, Diogo MM, Lobato da Silva C, Monteiro G, Grabherr R, Cabral JM: Plasmid DNA size does have an effect on nonviral gene delivery efficiency in stem cells. Cell Reprogram 2012, 14(2):130?37. 17. Wasley LC, Rehemtulla A, Bristol JA, Kaufman RJ: PACE/furin can method the vitamin K-dependent pro-factor IX precursor inside the secretory pathway. J Biol Chem 1993, 268(12):8458?465.More fileAdditional file 1: Primer sequences employed for cloning and sequencing the p 1.1 and p 1.two expression vectors. Abbreviations CHO cells: Chinese hamster ovary (CHO) cells; MTX: Methotrexate; eGFP: Enhanced green fluorescent protein; EBVTR: Concatemer of your terminal repeats from the Epstein-Barr virus; PCR: Polymerase chain reaction; Kbp: Kilo base pairs (1000 base pairs); EEF1A: Chinese hamster Elongation issue 1 alpha; IRES: Internal ribosome entry web site; DHFR: dihydrofolate reductase (EC 1.five.1.3); ORF: open reading frame; RFU: relative fluorescence units. Competing interests JAH, AGG and KGS declare that they’ve no competing interests. NAO, SVK and IIV are inventors from the patent RU2488633, which covers use in the p1.1 vector. Authors’ contributions NAO developed the experimental approach and plasmid design, performed cloning procedures, and drafted the manuscript. SVK performed the cell culture experiments and helped to draft the manuscript. IIV initiated the project, participated in its design and style and coordination and drafted the manuscript. NAO, JAH and IIV performed the copy quantity determination experiments, SVK, JAH and IIV performed eGFP level determinations. AGG and KGS coordinated the project. All the authors have study and authorized the final manuscript. Acknowledgments The authors wish to thank Dr Sergei Khaidukhov for performing the FACS evaluation and Dr Ivan Vorobiev (senior) for delivering the suggestions for FACS information interpretation. We also thank Dr Ivan Zvyagin, Lev Usakin, Maria Kordyukova and Victoria Shender for taking component in the molecular cloning procedures. The operate was supported by the following grants: RFBR 13-0440277- “Combinatorial chemistry and biology approaches to theranostics of various sclerosis”; RFBR 14-04-00647 ” Combinatorial biology approaches TXA2/TP Agonist Purity & Documentation making use of yeast show for improvement of biocatalysts de novo”; Program of your Presidium on the Russian Academy of Sciences No24 “Detection of Viral Antigens as you possibly can Trigger.