Pcmvtnt

Fragments encoding plasminogen K1-5 (PlgK1-5, amino acids 1-546) were generated and validated as described previously [8 (link)]. PlgK1-5 was then subcloned into the eukaryotic expression vector pCMVTNT (Promega, Mannheim, Germany) according to the manufacturer's protocol; correct insertion was determined through enzymatic restriction digestion and gel electrophoresis of pCMVTNT-K1-5 (pK1-5) on a 1% agarose gel. pCMVTNT without transgene (pMock) was used as a vector control.
pK1-5 and pMock were amplified using the E. coli K12 strain DH10B and plasmid vectors were isolated using the NucleoBond Xtra Maxi Plus EF system (Macherey-Nagel, Düren, Germany). DNA content and purity were determined by measuring the OD260/280 ratio.

Vector pCMVTNT-EPHX1 used for in vitro translation of microsomal epoxide hydrolase (mEH) was constructed by inserting of EPHX1 gene (GeneBank Accession No. NM_000120) cDNA into pCMVTNT (Promega, Madison, WI, USA) between the Xho I and Kpn I sites, EPHX1 gene cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the total RNA isolated from Huh-7 hepatoma cells. The primers used were: forward primer, 5′- CCGCTCGAGGCCACCATGTGGCTAGAAATCCTCCTCACT-3′; reverse primer, 5′-CGGGG TACCTCATTGCCGCTCCAGCAC-3′. pACT-EPHX1_353–455 coding for 353–455 amino acids of mEH was generated by an in-frame insertion of PCR amplified fragment using screened cDNA library prey plasmid as a template into pACT between the Sal I and Not I sites (encodes a herpes simplex virus type 1 VP16 protein, Promega). The primers used were: forward primer: 5′-ACGCGTCGACTTGACCTGCTGACCAAC-3′; reverse primer: 5′-ATAAGAATGCGGCCGC TCATTGCCGCTCCAGCAC-3′. pGEX-HBSP coding for GST-HBSP protein, pBIND-HBSP, pBIND-HBSP1-47 and pBIND-HBSP48-111, which respectively codes for GAL4 DNA-binding domain fused full length, N terminal 47 amino acids and C terminal 64 amino acids of HBSP, were described previously [21 (link)].

To construct expression plasmids for Myc-tagged PIG proteins in S2 cells, cDNAs encoding DmPIGs were obtained from the Drosophila Genomic Resource Center (DGRC) (DmPIG-B; LD47795) (DmPIG-L; GM02889) (DmPIG-M; GH02741) (DmPIG-V; RE16378) (DmPIG-X; IP05792) (DmPIG-N; AT21454) (DmPIG-F; AT13969) (DmPIG-O; SD07983). A DNA fragment encoding the 3Myc sequence was ligated to the 3′ ends of the PCR-amplified coding regions of DmPIG cDNAs and inserted into expression vector pRmHa. To construct the expression plasmid for Myc-tagged human PIG-B, cDNA tagged with the 3Myc epitope at the N-terminus was cloned into pRmHa. To generate chimeric PIG-B expression plasmids, different gene fragments amplified from DmPIG-B and human, ant, mosquito, bombyx or tribolium PIG-B were fused using the in-Fusion HD cloning kit (TAKARA) and inserted into pRmHa. To construct the expression plasmid containing Calr–GFP, the Calr–GFP gene fragment was amplified from ER–GFP (CA06507) fly cDNA and inserted into pRmHa. To construct expression plasmids in CHO cells, PCR-amplified fragments were inserted into expression vector pCMVTNT (Promega). To generate transgenic flies, PCR-amplified fragments were cloned into pJFRC4-3xUAS-IVS-mCD8::GFP. Plasmids were injected into y¹w⁶⁷c²³; P{CaryP}attP2 embryos and integrated into genomic site 68A4 using the phiC31 integrase system.