a Relative 5′RACE-Seq abundances of T7 promoter +2 to +8 sequence variants, grouped by +2/+3 dinucleotide. Promoters with three guanines at positions +1 to +3 (GGG) showed the highest activity on average (+1G is present in all tested promoter variants). 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. b Fraction of transcripts with the indicated +1 to +3 sequence, that have an additional G added to the 5′ terminus as a result of polymerase sliding during initiation. Shown are the values from two replicate 5′RACE-Seq experiments. c Comparison of the IVT activity of +4 to +8 T7 promoter variants with different 5′ RACE-Seq ranks. All T7 promoter variants comprised a GGG sequence at positions +1 to +3 and were used to in vitro transcribe a 410 nucleotides long RNA for 2 h. Shown is the fold amplification relative to the template DNA. Indicated below is the +4 to +8 RACE-Seq rank. c The highest ranked +4 to +8 sequence motifs were used for in vitro transcription with T7 polymerase. Shown is the resulting fold amplification of the template DNA after 1 h IVT. d The highest ranked +4 to +8 sequence motifs were used for in vitro transcription with T7 polymerase. Shown is the resulting fold amplification of the template DNA after 1 h IVT. e Comparison of the IVT activity of two T7 promoter variants using different IVT DNA template concentrations. IVT was performed for 2 h. All error bars represent standard deviation for triplicate experiments.