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Structure and Junction Analysis of DEN1 and DEN4 Chimeric Constructs

This document delves into the intricate structural and non-structural elements of chimeric constructs involving DEN1 and DEN4. It outlines the splice junctions created by various cut and ligation processes, utilizing enzymes such as Bgl.II, Pst.I, and Xho.I. The analysis includes sequence characterization, amino acid translations for multiple junctions, and the importance of linkers in maintaining the stability and function of constructs. This comprehensive examination aims to enhance our understanding of viral genome manipulation for biomedical applications.

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Structure and Junction Analysis of DEN1 and DEN4 Chimeric Constructs

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  1. A. Non-structural Structural C M E NS1 NS2A NS2B NS3 NS4A NS4B NS5 p4 (DEN4) 5’ 3’ DEN1 subclone pUCBXR-init Bgl II (Pst I) Xho I Chimerization 1 2 3 p4-D1L-CME 3’ 5’ Linker D30 p4D30-D1L-CME 3’ 5’ 5 4 p4-D1-ME 5’ 3’ D30 5’ p4D30-D1-ME 3’ B. DEN4 DEN1 Bgl II Junction 1: -CAGTTTGTTTGAATAGAGAGCAGATCTCTGgaaaaATGAACAACCAACGG- MetAsnAsnGlnArg- DEN1 Junction 2:-ATTATGAACAGGAGGAAAAAATCTGTGACCATGCTCCTCATGCTGCTGCCC- -IleMetAsnArgArgLysLysSerValThrMetLeuLeuMetLeuLeuPro- DEN1 DEN4 Xho I Linker Xho I Junction 3:-CTAGGATTAAACTCgagtagactaagttaactacctcGAGGAACACTTCA- -LeuGlyLeuAsnSer-----------------------ArgAsnThrSer- -LeuGlyLeuAsnSerSerArgLeuSer*** Virus, no linker: E. coli, with linker: DEN4 DEN1 Pst I Junction 4:-ATCTTGAACGGGAGAAAAAGGTCTGcagCCATGCTCCTCATGCTGCTGCCC- -IleLeuAsnGlyArgLysArgSerAlaAlaMetLeuLeuMetLeuLeuPro- DEN1 DEN4 Xho I Junction 5:-CTGCTGACATGGCTAGGATTAAACTCgAGGAACACTTCAATGGCTATGACG- -LeuLeuThrTrpLeuGlyLeuAsnSerArgAsnThrSerMetAlaMetThr-

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