a 5′RACE-seq using SP6 polymerase. Normalized average nucleotide composition from position +2 to +16 in RNA transcribed by SP6 RNA polymerase from a randomized SP6 promoter library. Substantial sequence preference was observed until the +3 nucleotide position. b Box plot showing relative abundances of +2 to +16 SP6 promoter variants detected in 5′RACE-seq, separated by +2/3 dinucleotide sequence. All variants have a G at +1. Promoters with +1 to +3 GAA showed highest activity. Each whisker plot represents 948–985 +1 to +8 motifs, dependent on homopolymer filtering. Whiskers reach to 1.5× IQR away from the 1st/3rd quartile. c IVT using high ranking +2 to +8 SP6 promoter variants with the indicated +2/3 dinucleotides. The +2/3 dinucleotide sequence appeared as main determinant of SP6 transcriptional activity. d IVT using SP6 promoter templates harboring +1 to +3 GAA followed by +4 to +8 sequence motifs of varying 5′RACE-seq rank. IVT was performed for 2 h. Shown is the resulting fold amplification of the template DNA. Sequence elements after +4 showed no effects on SP6 promoter activity. All error bars represent standard deviation for triplicate experiments.