Fig. 1

Development and validation of new pHybrid expression vector. (a) pHybrid-BFP-mScarlet vector map. Introduction of a new hybrid vector for bacterial and mammalian expression of target gene using mScarlet as reference gene. (b) pHybrid linear map showing basic features of pHybrid vector: (top) origin of replication, BFPs under CMV promoter and rhaB promoter for expression in mammalian cells and E. coli, respectively, followed by rrnB T1 terminator and SV40 poly-A tail, mScarlet under SV40 promoter regulation followed by HSV TK poly-A tail; (bottom) nucleotide sequence of the SD sequence, His-tag and Kozak sequence elements in front of the BFP gene. (c) Fluorescence images of E. coli bacteria transformed with pHybrid-mBlueberry2/mScarlet (mBlueberry2), pHybrid-EBFP2/mScarlet (EBFP2), and pHybrid-mTagBFP2/mScarlet (mTagBFP2) expression vectors in blue (top) and red (bottom) channels (imaging conditions: 403 nm/456 nm excitation/emission for blue, ex/em 561 mn/594 nm for mScarlet). (d) Fluorescence images of HEK cells transfected with pHybrid-mBlueberry2/mScarlet (mBlueberry2), pHybrid-EBFP2/mScarlet (EBFP2), and pHybrid-mTagBFP2/mScarlet (mTagBFP2) expression vectors in blue (top), red channel (middle) and merged (bottom) (imaging conditions: 403 nm excitation, 456 nm emission for BFPs; 561 nm excitation, 594 nm emission for mScarlet, 0.91 mW/mm² power). The dynamic range of all images was adjusted independently to facilitate visualization. (e) Correlation of HEK intracellular brightness and in vitro brightness for blue fluorescence variants selected from the random library of BFP derived from mRuby3 (see Supplementary Table ^S1 for statistics; Pearson’s correlation shown in graph; imaging conditions of HEK same as in (d)).