TRIM14 and HCV proteins-BiLC reporter constructs were cloned into the lentiviral vector backbone FG11F plasmid (Patent US20120201794 A1). GlucN (17-93aa) and GlucC (94-185aa) fragments (GenBank: AY015993) were inserted between the enzyme restriction sites AscI and RsrII of the FG11F vector. TRIM14 or HCV proteins were cloned into BiLC reporter system via the Gateway recombination cloning system (Invitrogen). HEK293T cells were seeded in 24-well plates the day before transfection. The lentiviral FG-EH-TRIM14-GlucN and FG-EH-HCV protein-GlucC plasmids were co-transfected into HEK293T cells, 48 hours later, the TRIM14 and HCV proteins-BiLC lentiviral particles were harvested, and luciferase activity was analyzed.
Gateway recombination cloning system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Gateway recombination cloning system is a technology developed by Thermo Fisher Scientific for efficient gene cloning. It allows for the rapid and reliable transfer of DNA sequences between multiple vectors. The system utilizes site-specific recombination to facilitate the movement of DNA fragments, enabling researchers to streamline their molecular biology workflows.
Automatically generated - may contain errors
Lab products found in correlation
Phusion high fidelity polymerase, by New England Biolabs (2 mentions)
Pdonr207, by Thermo Fisher Scientific (2 mentions)
Pog44 flp recombinase expression vector, by Thermo Fisher Scientific (2 mentions)
Pvsvg g, by Thermo Fisher Scientific (2 mentions)
Lr reaction, by Thermo Fisher Scientific (2 mentions)
Bp reaction, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Polyfect, by Qiagen (2 mentions)
Pspax2, by Addgene (2 mentions)
Gateway lr recombination reaction, by Thermo Fisher Scientific (1 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
8 protocols using gateway recombination cloning system
1
Bioluminescent Protein Interaction Assay
2
Gateway Cloning of CaMKII Isoforms
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To construct expression vectors, we routinely used the Gateway recombination cloning system (Invitrogen). The BP clones encoding human CaMKIIα (NM_015981.3, Addgene, Watertown, Massachusetts, USA, 23408), human CaMKIIβ (NM_172080.2, Addgene, 23820), human CaMKIIδ (NM_172127.2, Addgene, 23814), and human CaMKIIγ (NM_172170.5, Addgene, 23409) were purchased from Addgene. These genes were transferred from BP clones to several destination expression vectors, such as pDEST-HA-N, pDEST-Flag-N, and pDEST-mCherry-N by the Gateway LR recombination reaction (Invitrogen), according to the manufacturer’s guidelines.
3
Gateway Cloning of CRE Reporters
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CREs selected for analysis in transgenic reporter assays were cloned by PCR amplification of the fragment containing it plus flanking sequence from genomic DNA, using Phusion high fidelity polymerase (NEB). attB4 and attB1r sequences were included in the PCR primers for use with the Gateway recombination cloning system (Invitrogen). The amplified fragment was first cloned into the Gateway pP4P1r entry vector and sequenced using M13 forward and reverse primers for verification. In cases where a point mutation was engineered in the wildtype CRE, a site-directed mutagenesis kit (QuikChange II XL Site-Directed Mutagenesis Kit, Agilent) was used on the pP4P1r vector containing the wildtype CRE sequence. Test elements in the pP4P1r vector were combined with a pDONR221 construct containing either a gata2 promoter-eGFP-polyA or a gata2 promoter mCherry- polyA cassette, and recombined into a destination vector with a Gateway R4-R2 cassette flanked by Tol2 recombination sites [8 (link), 37 (link)]. The primer sequences used for amplification of the published CREs described in the manuscript are listed in S4 Table and images from independent lines for some of the constructs are presented in S1 Fig .
4
prp-31 expression and localization study
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The molecular construct to study the expression and localization of prp-31 (pCUC32) was made by the Gateway recombination cloning system (Invitrogen). The 5′ entry clone includes 664 bp of the promoter region, and 3′ entry clone contains the 77 bp of the 3′ UTR sequence (primer sequences available upon request). Plasmid pCM1.35 was used as the middle entry clone and pCFJ150 was the destination vector.
5
Retroviral Silencing of LTB and LTBR
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Retrovirus (pSI-MSCVpuro-H1R) particles carrying either shLTB or shLTBR and mock-infected controls were constructed using the Gateway recombination cloning system (Invitrogen, Waltham, MA, USA) as previously described [36 (link)]. HEK-293T cells were used for the production of these constructed retroviruses. Retroviral transductions were performed on HTK1-16E6SD, HTK1-LXSN, and SCC90 cell lines following a standard protocol. Briefly, the listed cell lines were cultured on 6-well plates for 24 h before infection. The retroviruses carrying shRNA were added into each culture well at the multiplicity of infection of 3 in the presence of 4 µg/mL of polybrene. After 24–30 h, cells were selected in the presence of 1 µg/mL of puromycin until the mock-infected cells completely died. The expression of LTB and LTBR was subsequently detected by quantitative RT-PCR. Either LTβ- or LTβR-silenced cells were cultured and used in the downstream experiments (Supplementary Table S5 ).
6
Cloning and Analysis of Lamprey Pax6β CNE
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An 878 bp fragment containing the CNE from the Japanese lamprey Pax6β locus plus flanking sequence was cloned by PCR amplification using Phusion high fidelity polymerase (NEB). attB4 and attB1r sequences (underlined in the primers below) were attached to the PCR primers for use with the Gateway recombination cloning system (Invitrogen). The amplified fragment was inserted into the Gateway pP4P1r entry vector using BP clonase and the sequence was verified using M13 forward and reverse primers. Primer sequences used for amplification of the lamprey CNE are:
Lamp6β_NRE_FP-B4:
5′-AACGGGGACAACTTTGTATAGAAAAGTTG GGAGATCGTGATGGAGGTGT-3′ and Lamp6β_NRE_RP-B1r:
5′-AACGGGGACTGCTTTTTTGTACAAACTTG ACCCCACGTGTACCGTCTAA-3′ Next, the lamprey CNE-containing pP4P1r entry construct was mixed with a pDONR221 construct containing a gata2 minimal promoter-eGFP-polyA cassette, and recombined using LR Clonase into a destination vector with a Gateway R4-R2 cassette flanked by Tol2 recombination sites to produce the LjPax6β-CNE-gata2-eGFP reporter construct. The minimal gata2 promoter-eGFP reporter cassette has been used to report on the tissue-specific expression patterns driven by a wide variety of linked enhancers and does not produce reporter expression without the presence of linked enhancer elements57 (link).
Lamp6β_NRE_FP-B4:
5′-AAC
5′-AAC
7
Recombinant Protein Production and Cell Line Engineering
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For cloning the Gateway recombination cloning system (Invitrogen) was used. p27 mutants were generated by site-directed mutagenesis. In BP reactions (Invitrogen) PCR products were recombined with the pDONR207 (Invitrogen) vector. Entry vectors were recombined with destination vectors carrying the desired tags (LR reactions, Invitrogen). To generate stable cell lines, pTO destination vectors (generated by Stephan Geley) were co-transfected with the Flp-recombinase expression vector pOG44 (Invitrogen). To lentivirally transduce p27−/− MEFs and MDA-MB-231 cells, the plasmids pHR-SFFV-IR-puroSG (a generous gift from Stephan Geley), pVSVG-G (Invitrogen) and psPAX2 (Addgene, Cambridge, MA, USA) were used. pET3c-p27 (wild-type) was used to produce recombinant untagged human p27. Murine p27 was cloned into pET21eSG (a generous gift from Stephan Geley) for expression in E. coli. Rho binding domain of rhotekin was expressed in E. coli using pGEX-2 T-RBD. For transfection Lipofectamine 2000 (Invitrogen) or PolyFect (Qiagen, Venlo, Netherlands) were used.
8
Cloning and Expression of p27 and RBD
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