Clonexpress ultra one step cloning kit

Mouse Cfap52 was obtained from mouse testis cDNA and cloned into the pEGFP-C1 vector using the Clon Express Ultra One Step Cloning Kit (C115, Vazyme). Mouse Cfap45 was obtained from mouse testis cDNA and cloned into the pCMV-Myc vector using the Clon Express Ultra One Step Cloning Kit (C115, Vazyme).

The full-length sequence of slc45a2 was amplified from cDNA derived from WT C. argus via PCR using the following primers: slc45a2-HindIII F: 5′-ctagcgtttaaacttaagcttATGACCCTGTTATCAGAGGACCA-3′ and slc45a2-BamHI R: 5′- ccacactggactagtggatccTCAATCCACATATCTGACAAAGAGG-3′. The product was ligated into the pcDNA3.1 (+) vector using the ClonExpress® Ultra One Step Cloning Kit (Vazyme, China). The resulting plasmid was verified by sequencing, and messenger RNA was transcribed using the T7 High Yield RNA Transcription Kit (Vazyme, China). Injections were carried out at 1–4 cell stage of YM snakehead at a concentration of 200 ng µl⁻¹. After injection, embryos were incubated at 26°C until hatching. The phenotype of injected embryos was observed from 24 hpf and photographed by an SZ810 stereomicroscope (CNOPTEC, China).

Standard molecular cloning protocols were used to construct the plasmids used in this study. Q5^® High-Fidelity DNA Polymerase (NEB) was used for PCR amplifications and E. coli DH5α was used for plasmid preparation. The construction of the Int expression vectors (pCMVssInt-h/218 and -Int-C3) has been described previously (Vijaya Chandra et al., 2015 (link)). pEF_attP_mCherry was prepared by replacing the Neo_IRES_dTomato cassette with the mCherry_loxP cassette (PCR amplified using mCherry_BamHI and SV40_LoxPmCherry_HindIII primers listed in Supplementary Table S1) in pEF_attP_Neo_IRES_dTomato between the BamHI and HindIII sites.
The pattB_HygroR_eGFP plasmid was constructed by cloning the eGFP expression cassette into the NaeI site in pattB_HygroR by homologous recombination cloning using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China) using the manufacturer’s protocol. The pattB_HygroR_PD1 HC and LC_PuroR_eGFP plasmids were constructed and obtained from e-Zyvec (Loos, France).

The nisI fragment was obtained from pet30a-nisI plasmid (Synthesized by Shanghai Personal Biotechnology Co., Ltd) by PCR with the 5′ and 3′ terminal containing homologous arm sequences identical to the pMG36e plasmid inserted site (Table 1). Similarly, the linear fragments without erythromycin resistant gene was acquired from the pMG36e-lacLM and pMG36e-lacZ plasmid by reverse PCR and the primers also were showed in Table 1. Then, the nisI fragment and the above linear fragments were homologous recombined by ClonExpress Ultra One Step Cloning kit (Vazyme, China). These new plasmids were designated as pMG36n-lacLM and pMG36n-lacZ, respectively (Fig. 1).

The full-length sotB and lapC genes from the E. coli MG1655 genome were subcloned into the cloning vector pET28a, together with the fusion of a C-terminal 8 × His tag. Site-directed variants were produced by the ClonExpress Ultra One Step Cloning Kit (Vazyme Biotechnology Co., Ltd., Nanjing, China) with the plasmid pET28a/sotB as the template and verified for fidelity by DNA sequencing in Tsingke Biotechnology Co., Ltd. (Beijing, China).

All
primers used in this study are listed in Table 2. Three different ptxD genes
from P. stutzeri,^{22 (link)}P. aeruginosa,^{27 (link)} and K. pneumoniae(28 (link)) were tried. The ptxD genes were expressed based on the pXMJ19 plasmid under the Peftu promoter. The Peftu promoter were
amplified from the pJYS3_crtYf plasmid using primers
*Peftu-F/*Peftu-R. The ptxD_Pst, ptxD_Pae, and ptxD_Kpn gene fragments (with
sequences homologous to plasmid pXMJ19) were codon-optimized and synthesized
by Nanjing Tsingke Biological Technology Co., Ltd. The Peftu promoter and each of the ptxD genes were ligated
into BamHI-linearized pXMJ19 plasmid, generating recombinant plasmid
pXMJ19*ptxD_Pst, pXMJ19*ptxD_Pae, and pXMJ19*ptxD_Kpn, respectively. The ligation
was accomplished through homologous recombination using the ClonExpress
Ultra One Step Cloning Kit (Vazyme) according to the manufacturer’s
protocol.

For the construction of plasmids overexpressing WT BMI1 and its nuclear localization signal (NLS2) mutant (BMI1-△NLS2), mouse Bmi1 cDNA was PCR-amplified and then cloned into the pcDNA3.0 vector (Invitrogen, Carlsbad, CA, United States). For the construction of pGL6-Map3k3 plasmids, both full length (−2000 ∼ + 100 relative to the transcriptional start site [TSS]; promoter-1) and partial (−1033 ∼ + 100, promoter-2 and −662 ∼ + 100, promoter-3) Map3k3 promoter sequences were PCR-amplified from mouse genomic DNA and inserted into the pGL6 firefly luciferase reporter vector (Beyotime, Nantong, China). Both the BMI1-△NLS2 and Map3k3 promoter mutant constructs were generated using the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). Bmi1 siRNA (5′-ACAAUGAAAGUUAAAAGUCGU-3′), Map3k3 siRNA (5′-AGUCUAAUGCCUCUUGUUCAU-3′), and negative control (NC) siRNA (5′-ACGUGACACGUUCGGAGAA-3′) were synthesized by GenePharma (Shanghai, China).
The transient transfection of expression plasmids, luciferase reporter plasmids, or siRNAs was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The transfected plasmids and siRNAs are indicated in the figures and figure legends. NC siRNA and empty vectors (EVs) were used as negative controls. After transfection, cells were utilized for measurements at the times indicated in the figure legends.