Pal ta

Purification of PCR products and products of digestion was performed by gel electrophoresis and subsequent extraction with the Cleanup Standard Kit (Evrogen, Moscow, Russia). The cDNA fragments of COL7A1 from FEB cells were cloned into the pAL-TA (Evrogen, Moscow, Russia), and the plasmids were used for Sanger sequencing (Evrogen, Moscow, Russia).

The region between the markers D17Mit21 and 22 contains the following genes: H2-Ab1, H2-Aa, H2-Eb1 and H2-Eb2. To clone the H2-Ab1 and H2-Aa genes, RNA was extracted from spleens using SV Total RNA Isolation System (Promega, Madison, WI, USA) and treated with DNase I (AMPD1,Sigma-Aldrich, St.Louis, USA). cDNA was synthesized with oligo-dT18 primers (Thermo Fisher Scientific, Inc) and M-MLV reverse transcriptase (Promega, Madison, WI, USA). Primer sequences for cloning were selected from the Ensembl database (version GRCm39) for the H2-Ab1, H2-Aa genes of C57BL/6 mouse strain. 5’ forward primers ended up at the ATG start codon; reverse primers started at the TGA stop codon. Coding DNA was amplified using Advantage GC Genomic LA Polymerase (Clontech, Takara Bio, USA, Inc). PCR products were extracted from gels with Cleanup Mini Set (Evrogen, Moscow, Russia) and cloned in the PCR-Script Amp Cloning vector using the PCR-Script™ Amp Cloning Kit (Agilent (Stratagene, Santa Clara, CA,USA) or pAL-TA (Evrogen, Moscow, Russia) after 3 preliminary cycles of PCR products amplification with Taq-polymerase (Helicon, Moscow, Russia). Four-five positive clones were sequenced for each gene.

LysECD7 protein (NCBI AN: YP_009602067.1) was obtained as described before [20 (link)], synthetic gene was used for investigations. Briefly, LysECD7′s initial coding sequence was artificially synthesized in a pAL-TA commercial vector (Evrogen Ltd., Moscow, Russia). Thereafter endolysin ORF was amplified from a pALTA-LysECD7 clone and integrated into the expression vector pET-42b(+) (Evrogen Ltd., Moscow, Russia), resulting in a pET42b-LysECD7-8his plasmid. All constructs were checked for errors via Sanger sequencing.