Nebuilder hifi dna assembly cloning

To make an exact copy of the 94 AA protein (SNZR P), an Impact kit (New England BioLabs, Ipswich, MA, USA) was used with the following modifications. The vector pTYB21 was used so that an N-terminus methionine would not be added to SNZR P. Because the cDNA encoding the 94 AA protein contains Sapl restriction enzyme site, Sapl could not be used in the cloning. Instead, another kit was used, NEBuilder HiFi DNA Assembly Master Mix/NEBuilder HiFi DNA Assembly Cloning (New England BioLabs, Ipswich, MA, USA) that allows assembly of the vector to SNZR P without the need for restriction enzymes. Another change was to transform the E. coli strain DH5a first and then subcloned into T7 express (New England BioLabs, Ipswich, MA, USA). The induction of these cells used 0.4 mM IPTG at 30 °C for 5 h. The sonicator (diagenode, Denville, NJ, USA) used to break open the E. coli was set at 4 °C at high power with 20 cycles of 30 s on/30 s off. The other directions in the kits were closely followed.

Full-length COL7A1^c.425A>G was cloned by the Golden Gate cloning technique using NEBuilder^® Hifi DNA Assembly Cloning. The whole 9.2 kB cDNA was split into two fragments (F1: 4461 bp; F2: 4440 bp) and cloned into a pMXs-IRES-Blasticidin Retroviral vector (Cell Biolabs, San Diego, CA, USA) (F3: 5636 bp). Primers were designed using the NEBridge ^® Golden Gate Assembly Tool. Amplification of the fragments was performed using Phusion^TM High-Fidelity DNA Polymerase (ThermoFisher Scientific, Waltham, MA, USA) for F1 at an annealing temperature of 63.4 °C (forward primer: 5′-cctcgagggccggcgcgccgcATGACGCTGCGGCTTCTG-3′; reverse primer: 5′-cctttggggccAATAGCTCCAGGAGGTCCC-3′) and for F2 at an annealing temperature of 66.2 °C (forward primer: 5′-ctggagctattGGCCCCAAAGGTGACCGG-3′; reverse primer: 5′-gaggggcggaatttacgtagcTCAGTCCTGGGCAGTACCTG-3′). The vector was linearized with NotI restriction enzyme (New England Biolabs, Ipswich, MA, USA) and cloned according to the NEBuilder^® Hifi DNA Assembly Cloning (New England Biolabs, Ipswich, MA, USA) instructions.

The position of protein helix inside, transmembrane, and outside of the endoplasmic reticulum (ER) were predicted using in silico transmembrane helix prediction (see foot note 3) (Supplementary Figure 1). In this study, the N-terminal domain are represented by the protein located inside ER together with the transmembrane helix. To swap the N-terminal domain of MtCPR1 and MtCPR2, the first 46 amino acids of MtCPR1 are swapped with the first 60 amino acids of MtCPR2 using NEBuilder^® HiFi DNA Assembly cloning to produce N-terminal sequence of MtCPR1 fused with truncated-N-terminal MtCPR2 (M1N-M2C) and N-terminal sequence of MtCPR2 fused with truncated-N-terminal MtCPR1 (M2N-M1C). Two different fragments of each designed chimeric CPRs (M2N-M1C and M1N-M2C) were amplified using pENTR-MtCPR1 and pENTR-MtCPR2 as template and primers listed in Supplementary Table 2. A minimum of 20-bp overlapping nucleotides of each CPR mutant and pENTR™ as vector backbone were designed for fusion cloning.