Purelink hipure expi plasmid megaprep kit

The pcDNA3.1(+) plasmid was used as a template for inserting the MUC2-C (NCBI reference: MH593786.1^{8 (link)} (protein AZL49145.1) containing the amino acids 4356–5130 with an N-terminal IGk signal sequence, a 6 His-tag and the 3C precision cleavage tag in that order from the N-terminal. The mammalian expression vector was transformed through electroporation using XL1 Blue Electrocompetent cells (Agilent). The transformed cells were selected with the corresponding antibiotic and grown overnight in LB Broth Base (Invitrogen). DNA was prepared using PureLink™ HiPure Expi Plasmid Megaprep Kit (Thermo Fisher Scientific).

Gene expression vectors were constructed either by employing restriction endonucleases (New England Biolabs, Ipswich, MA, USA) followed by ligation using T4 DNA ligase (New England Biolabs, cat. no. M0202L) or by Gibson assembly (New England Biolabs, cat. no. E2611L). For restriction enzymes‐based cloning, digested plasmid backbones were dephosphorylated with antarctic phosphatase (New England Biolabs, cat. no. M0289L) before ligation. PCR reactions were performed using either Phusion High‐Fidelity DNA polymerase (New England Biolabs, cat. no. M0530L) or Q5 High‐Fidelity DNA polymerase (New England Biolabs, cat. no. M0491L). For Gibson assembly, the PCR products were amplified using primers that had 15–20 bp complementary sequences at each end. Detailed information about restriction enzymes and primers used for the design of each plasmid is presented in Table S1, Supporting Information. Plasmids were transfected and propagated in XL10‐Gold ultracompetent Escherichia coli (New England Biolabs, cat. no. C2992) and DNA was extracted using a plasmid mini‐prep kit (Zymo Research, Irvine, CA, USA, cat. no. D4054) or a ZymoPURE II Plasmid Midiprep Kit (Zymo Research, cat. no. D4200). A PureLink HiPure Expi plasmid megaprep kit (Thermo Fischer Scientific, Waltham, MA, USA, cat. no. K210008XP) was used for larger‐scale preparation.