P gemt easy vector a1360

The MgCh sequence in sugarcane was compared to sorghum and maize MgCh sequences via tBLASTn. This allowed the conserved domains to be identified, informing the primer design for the PCR amplification of multiple allelic MgCh variants from sugarcane target cultivar CP88-1762 (WT) (Supplementary Table 1). The amplicons were cloned into the p-GEMT® easy vector (A1360) (Promega, WI, USA), followed by the Sanger sequencing of multiple colonies. The sgRNAs were selected in silico using CRISPOR (http://crispor.tefor.net/). sgRNA1 was designed to cleave to a highly conserved region, while the sgRNA2 target was less conserved and included three allelic variants that differed in the number of mismatches (0, 1, or 2) in the genomic target sequence of the sgRNA (Figure 2A).

A search for the hemp subtelomeric repeat was performed with a BLAST search (http://blast.ncbi.nlm.nih.gov) using the hop subtelomeric repeat sequence (GU831574) to probe the Cannabis sativa whole-genome shotgun contigs. In the search results, a contig (AGQN01004814) with one repeat of low homology to the hop subtelomeric tandem repeat was found. Nineteen tandemly repeated units about 375 bp in size from this contig were found using the GenDoc software (http://gendiapo.sourceforge.net) and were subsequently used for primer design. The primers (CS-1f 3′-GGTACCACTATGAGAAATGTGAGA-5′ and CS-1r 3′-CCTTTGTGAAATGTGGCCC-5′) used to amplify the detected repeat units were designed using PRIMER3 v.0.4.0 (http://frodo.wi.mit.edu). The PCR products from the CS-1 reaction were purified and cloned into the pGEM®-T Easy Vector A1360 (Promega, USA) in E. coli cells. Plasmids with inserts were isolated from cells using the GeneJET ™ Plasmid Miniprep Kit (Fermentas, Lithuania). The nucleotide sequences of the inserts were determined on an ABI3130XL instrument (Applied Biosystems, Inc., USA) after sequencing reactions were performed with a Big Dye Terminator v 1.1. Cycle Sequencing Kit (Applied Biosystems, Inc., USA).