Epitope tags of the Bacillus BioBrick Box. (A) Pre- and suffixes of different cloning standards. The start- and the first stop-codon are underlined. RFC10  describes the commonly used BioBrick standard. To combine two parts, the upstream part is cut with SpeI, the downstream one with XbaI (red sites). The compatible overhangs of the two isocaudomers can be ligated, and result in an eight basepair scar that cannot be recut but is optimal for RBS spacing. This causes a frameshift that is not suitable for translational fusions. RFC25  describes the “Freiburg standard” which was developed by the 2007 iGEM-Team from Freiburg to allow translational fusions. Two parts can be combined similar to RFC10, but using NgoMIV and AgeI as restriction enzymes. The resulting scar ACCGGC codes for Threonine and Glycine. RFC25M refers to our modification of RFC25 by adding an optimized RBS (yellow). While this convenient modification simplifies the cloning procedure, it should be noted that as a consequence it also prevents downward compatibility, since the resulting BioBrick directly adds two functional DNA parts in a single step. The tag-BioBricks and gfpmut1 are designed in that standard. (B) Nucleotide and amino acid sequences of the epitope tags, without the RFC25M pre- and suffixes shown in (A). (C) Western blots of N- and C-terminal fusions of each tag to GFP, using the strains TMB1920 (Flag-gfp), TMB1921 (gfp-Flag), TMB1922 (HA-gfp), TMB1923 (gfp-HA), TMB1924 (cMyc-gfp), TMB1925 (gfp-cMyc), TMB1926 (His-gfp), TMB1927 (gfp-His), TMB1928 (StrepII-gfp) and TMB1929 (gfp-StrepII). For each construct, two independent clones were tested with epitope tag- and GFP-specific antibodies as a positive control.