design: The ribozyme library was prepared using run off in vitro transcription using T7 polymerase. DNA templates for transcription were generated by PCR amplification of a gBlock (ordered from IDT DNA) containing the trans-splicing ribozyme sequence with an internal guide sequence (IGS; 5'-GACATA-3') complementary to JAK2-V617F at its G>T variant site (3'-TTGTAT-5') and a 30 nucleotide long 3'-tail (26). The gBlock was amplified using a 3'-PCR primer encoding the barcode library. This primer contained 25 nucleotides complementary to the ribozymes 3'-tail and added a partially randomized barcode as well as a 19 nucleotide long constant region (Fig. 1). Six different 5'-PCR primers, each containing a T7 promoter sequence and a randomized nucleotide region, were used to append six different EGS library designs at the ribozyme 5'-terminus. The randomized region was 13 (N13), 15 (N15), or 18 (N18) nucleotides long. N13 primers contained an upstream region that was designed to form an 'antisense duplex' with the substrate in one of four different registers (R+3, R+4, R+5, or R+6). Together, this resulted in six distinct sub-libraries (see SM, Section 1 for gBlock and all primer sequences). All PCR products were purified using PCR clean up columns (Macherey-Nagel, Dren, Germany) and their concentration was measured using a Nanophotometer (Implen, Mnchen, Germany). The six sublibraries were mixed in equimolar amounts and diluted to a final concentration of ~108 molecules per 10 L. PCR amplification of 10 L of this solution resulted in a 'constrained complexity library' with thousands of copies from each of about 108 different sequences.