Coding sequences of mouse full length Suv420h2, or of the SET domain, or clamp domain of Suv420h2 were cloned into pEGFP (addgene), pEF4 (Invitrogen), and pGEX6P1 vectors in order to generate GFP-tagged, FLAG-tagged full length, SET domain, or clamp domain versions of Suv420h2, respectively. Also, full length of Suv420h2 was cloned into pAM_1C vector (Active motif) by In-Fusion HD Cloning kit (Takara) and then AM-tagged Suv420h2 full length was cloned into pGCDN-IRES/EGFP retroviral vector by using restriction enzyme site of NotI and XhoI. For HP1α, cording sequence of mouse HP1α was cloned into pEF4 vector by using restriction enzyme site of EcoRI and XbaI. pGEX6P1 HP1α was generated by In-Fusion HD Cloning kit (Takara). Other pGEX6P1 constructs including pGEX6P1 PR8-PB2, pGEX6P1 PR8-NP, pGEX6P1 PR8-M1, and pGEX6P1 PR8-NS1 as well as GFP-tagged HoxC8 (pEGFP+HoxC8) were generated by In-Fusion HD Cloning kit (Takara). Primer sequences used for all cloning constructs were listed in Table S1 .
In fusion hd cloning kit
Manufactured by Takara Bio
Sourced in United States, Japan, China, France, Germany, Canada
The In-Fusion HD Cloning Kit is a versatile DNA assembly method that allows for the rapid and precise seamless cloning of multiple DNA fragments. The kit provides a high-efficiency, directional cloning solution for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Stellar competent cells, by Takara Bio (26 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (25 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (19 mentions)
Cloneamp hifi pcr premix, by Takara Bio (17 mentions)
Primestar gxl dna polymerase, by Takara Bio (17 mentions)
Primestar max dna polymerase, by Takara Bio (17 mentions)
Primestar hs dna polymerase, by Takara Bio (15 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (14 mentions)
Primestar mutagenesis basal kit, by Takara Bio (13 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (12 mentions)
Dual luciferase reporter assay system, by Promega (11 mentions)
Qiaquick gel extraction kit, by Qiagen (11 mentions)
Geneart, by Thermo Fisher Scientific (10 mentions)
Kod plus neo, by Toyobo (10 mentions)
Pcdna3, by Thermo Fisher Scientific (9 mentions)
Qiaprep spin miniprep kit, by Qiagen (9 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (9 mentions)
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Smarter race 5 3 kit, by Takara Bio (7 mentions)
Kod fx neo, by Toyobo (7 mentions)
1 385 protocols using in fusion hd cloning kit
1
Cloning and Tagging of Suv420h2 and HP1α
2
Donor Plasmid Construction for Plasmodium Reporters
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Donor plasmids used in this study are shown in Supplementary Fig. 2a . The DNA sequence of P. falciparum codon-optimised NanoLuc fused with T2A and part of the GFP ORF (the complete sequence is shown in Supplementary Fig. 2b ) was synthesised by Integrated DNA Technologies (IDT, gBlocks®). To construct the donor plasmid harbouring the GFP-T2A-NanoLuc expression cassette, we first PCR-amplified (KOD Hot Start DNA Polymerase, Merck Millipore) the sequence containing partial GFP, T2A, and full NanoLuc using primers P9/P10 (Supplementary Table 1 ). The amplified GFP-T2A-NanoLuc sequence was introduced into the BtgZI/AvrII site of pLf0127 (p47 gRNA/p47 HR1-HR2 GFP) using the InFusion reaction (In-Fusion® HD Cloning Kit; Clonetech), which was previously used to generate a transgenic line expressing GFP16 (link). To construct the mCherry-T2A-NanoLuc expression cassette, we PCR-amplified the T2A-NanoLuc using primers P11/P10 (Supplementary Table 1 ). The amplified PCR fragment was introduced into the EagI/AvrII site of pLf0128 (p47 gRNA/p47 HR1-HR2 mCherry-Luc) by an InFusion reaction (In-Fusion® HD Cloning Kit, Clontech) to replace firefly luciferase with the T2A-NanoLuc coding sequence. The donor/gRNA plasmid for mCherry-Luc (pLf0128) has been described previously16 (link).
3
Generation of Optogenetic Tools for Neuroscience
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To generate pAAV-EF1a-DIO-hOPN4-mCherry, pAAV-EF1a-DIO-hM3Dq-mCherry (addgene#50460) was modified. The coding sequences of hM3Dq was replaced by the sequence coding human OPN4 isoform1 (Genbank: NM_033282) in pAAV-GFAP104-melanopsin-mCherry (addgene#122630).50 (link) Amino acid from 385 to 478 and from 400 to 478 was deleted for hOPN4-dC and hOPN4-9A-dC, respectively. For hOPN4-9A and hOPN4-9A-dC, synthesized oligo nucleotides with mutations from serine/threonine to alanine (SA/TA) were inserted into pAAV-EF1a-DIO-hOPN4-mCherry by In-Fusion HD Cloning Kit (Z9648N, Clontech Laboratories Inc.) at the position of amino acid 384, 387, 388, 391, 392, 394, 395, 397 and 398. pEF1α-hOPN4-mCherry and pEF1α-hOPN4dC-mCherry was generated by insertion of the coding sequences of hOPN4-mCherry and hOPN4dC-mCherry into pEF1α-mCherry-N1 (Clontech Laboratories Inc) by In-Fusion HD Cloning Kit (Z9648N, Clontech Laboratories Inc). pGL4.30-NFAT-Luc vector was purchased ([luc2P/NFAT-RE/Hygro], E8481, Promega Corp.).
4
Cloning and Transformation of Yeast Plasmids
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Yeast plasmids have a pRS backbone with centromeric replication site18 (link) and were cloned with homologous recombination in S. cerevisiae or with the Infusion HD cloning kit (Clontech, USA) in E. coli. All FAS1 and FAS2 derived constructs carry the native promoter and terminator sequences19 (link). Yeast transformation was done with the LiOAc-method44 (link). E. coli plasmids have a pET22b backbone (Novagen, USA) and were cloned with the Infusion HD cloning kit (Clontech, USA) (see Table S1 ).
5
CRISPR/Cas9-mediated TK1 Knockout
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To knock-out TK1 using the CRISPR/ Cas9 genome editing system, guide RNA (gRNA) sequences were designed using the online software CRISPRdirect25 (link). The targeting sequences of gRNAs are provided in Fig. 1A and Supplementary Table 3 . The sense and antisense oligonucleotides were annealed and cloned into the BbsI site of pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene plasmid #42230), which was a gift from Feng Zhang26 (link). To construct donor vectors by PCR, template DNA (genomic DNA of HCT 116 cells) was amplified using KOD FX DNA polymerase (TOYOBO) and primers containing a sequence homologous to the target locus, which are provided in Supplementary Table 3 . The amplified left- and right-arm DNA fragments, which contained approximately 600–700 base pairs homologous to the target locus, and a puromycin resistance cassette were cloned into the BamHI–NotI site of pBluescript SK+ using an In-Fusion HD Cloning Kit (Takara-Clontech). To construct the stable and inducible TK1 expression plasmids, the TK1 gene was amplified from template DNA (total cDNA of HCT 116 cells) using KOD FX DNA polymerase (TOYOBO) and primers (Supplementary Table 3 ), and then cloned into the pcDNA3.1 (Thermo Fisher Scientific) and pTetOne (Takara-Clontech) vectors using an In-Fusion HD Cloning Kit (Takara-Clontech), respectively.
6
Generation of B7H7 and CD28H Constructs
7
Fluorescent Protein Tagging of Arabidopsis Proteins
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To generate PICALM5a-GFP, PICALM5a-mRFP, PICALM5b-GFP, GFP-SYP124, GFP-SYP125, and GFP-SYP131, approximately 2 kb of upstream sequences and 1 kb of downstream sequences for the coding regions of each gene were PCR-amplified with the primers listed in Supplementary Table 2 . The amplified fragments were then cloned into the pENTR/D-TOPO entry vector (Thermo Scientific). The clones were amplified by inverted PCR and combined with cDNA for GFP or mRFP using an In-Fusion HD Cloning Kit (Clontech).
To generate ANX1-GFP and ANX2-GFP, PCR-amplified genomic fragments containing approximately 2 kb of promoter sequences and coding regions for ANX1 and ANX2 were cloned into the pENTR/D-TOPO entry vector. The clones were recombined with the pGWB4 vector34 (link) using an LR Clonase II enzyme mix (Thermo Scientific).
To generate LAT52p:CLC1-GFP, a genomic fragment containing the coding region for CLC1 without the stop codon was PCR-amplified and subcloned into the SpeI site of the YMv036 vector35 (link) using an In-Fusion HD Cloning Kit (Clontech). PRK6-mClover was described previously20 (link).
To generate ANX1-GFP and ANX2-GFP, PCR-amplified genomic fragments containing approximately 2 kb of promoter sequences and coding regions for ANX1 and ANX2 were cloned into the pENTR/D-TOPO entry vector. The clones were recombined with the pGWB4 vector34 (link) using an LR Clonase II enzyme mix (Thermo Scientific).
To generate LAT52p:CLC1-GFP, a genomic fragment containing the coding region for CLC1 without the stop codon was PCR-amplified and subcloned into the SpeI site of the YMv036 vector35 (link) using an In-Fusion HD Cloning Kit (Clontech). PRK6-mClover was described previously20 (link).
8
Generation of Recombinant LSD1 and GSK3β Constructs
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Full-length and truncated LSD1 constructs were generated as described previously54 (link). HA- GSK3β was a gift from Dr. Chi-Neu Tsai (Chang-Gung University, Taoyuan, Taiwan).The pNTAP-GSK3β deletion constructs were amplified and cloned into pNATP vector using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA). The double-mutant (S707A/S711A) LSD1 and HA-GSK3β S9A were generated by overlapping PCR with a Q5 Site-Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer’s protocols. To construct the expression vector for the stably expression in cancer cells, the PCR products were amplified from Flag–LSD1 and Flag–LSD1 S707A/S711A and cloned into pLAS5w.PeGFP-I2-Puro (RNAi core laboratory, Sinica, Taiwan) with In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s protocols. The primers used in DNA constructs are shown in Supplementary Table 1 . The procedure was confirmed by DNA sequencing.
9
Engineered Mammalian Expression Plasmids
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To generate the pTOMM20-N-10-CLIPf mammalian expression plasmid, the DNA of CLIP tag was PCR amplified from pCLIPf vector (plasmid source: the Michael Davidson Fluorescent Protein Collection at the University of California, San Francisco (UCSF) Nikon Imaging Center) using primers (forward: 5′-GCGGGGATCCACCGGTCGCCACCATGGACAAAGACTGCGAAATGAAGC-3′; reverse: 5′-TCTAGAGTCGCGGCCGCTTAACCCAGCCCAGGCTTGCCC-3′). We then performed restriction enzyme digestion on vector pmEmerald-TOMM20-N-10 (plasmid source: the Michael Davidson Fluorescent Protein Collection at the UCSF Nikon Imaging Center), cutting out the mEmerald sequence between BamHI and NotI. The PCR-amplified CLIP tag was then ligated with the digested vectors using the In-Fusion HD Cloning kit (Clontech). The plasmids pSNAPf-Clathrin-15 and pSNAPf-LMNA were directly purchased from UCSF Nikon Imaging Center. For constructing the lentiviral production vectors, DNAs of TOMM20-N-10-CLIPf and SNAPf-Clathrin-15 were directly PCR amplified from mammalian expression constructs and subcloned into lentiviral pHR-SFFV vector (BamHI/NotI) using the In-Fusion HD Cloning kit (Clontech).
10
Engineering CAPTURE Vectors for Targeted Genome Editing
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To generate the pLVX-EF1a-BirA-P2A-FB-dCas9-IRES-zsGreen1 (CAPTURE1.1) vector, the BirA-V5-6xHis and FB-dCas9 were amplified from the pEF1a-BirA-V5-neo vector (Addgene, #61357) and pEF1a-FB-dCas9-puro (Addgene, #100547) as templates, respectively, and cloned into XbaI-digested pLVX-EF1a-IRES-zsGreen1 by In-Fusion® HD Cloning Kit (Clontech). To construct the pLVX-EF1a-NBio-dCas9-IRES-zsGreen1 (CAPTURE2.0-NBio) and pLVX-EF1a-dCas9-CBio-IRES-zsGreen1 (CAPTURE2.0-CBio) vectors, the dCas9 and BioTAP sequences were amplified from the pEF1a-FB-dCas9-puro (Addgene, #100547) and pEF1a-FB-dCas9-bioTAP-puro, respectively. Then the PCR amplicons were subcloned into XbaI-digested pLVX-EF1a-IRES-zsGreen1 by In-Fusion® HD Cloning Kit (Clontech). The pLVX-EF1a-BirA-P2A-FB-dCas9-IRES-zsGreen1, pLVX-EF1a-NBio-dCas9-IRES-zsGreen1, and pLVX-EF1a-dCas9-CBio-IRES-zsGreen1 vectors were deposited to Addgene under #138417, #138418, and #138419, respectively.
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