Clone-it Cloning Systems

Instant and Accurate High-throughput Cloning into Lentivectors

 

Features and Benefits of the Clone-it™ Lentivector Systems

cDNA Cloning System Details

Seamlessly clone your cDNA without the worries of internal restriction enzyme sites using SBI's Clone-it cDNA vectors.  This novel system enables the rapid cloning of any cDNA up to 5kb.  Highly active EF1α promoter drives transcription, and Kozak elements are built-in for optimized translation.  Proteins can optionally be expressed with N-terminal HA epitope or C-terminal T2A tag.  RFP reporter (Cat# LF520A-1) or Puromycin resistance gene (Cat# LF521A-1) are coexpressed for sorting or selection.

 

Clone-it cDNA Vector Map Download Vector Sequence: LF520A-1 • LF520B-1 • LF521A-1	Cloning Efficiency Data
Accurate & Directional Cloning	Monitor positive expressing  cells using RFP reporter
Easily create stable cell lines expressing your cDNA. RFP Cell Sorting or Puromycin Selection.	The cDNA constructs can be used in transient transfection experiments (shown left) or can be used to package lentiviral particles. The particles can then be used to transduce any cell type to create stable cell lines permanently expressing the protein of interest. Examples of VSV-g pseudotyped lentiviral transductions are also shown.

Details of the Lentiviral Delivery System

shRNA Cloning System Details

shRNA cassette can be directly assembled from short, tagged oligos and mixed with ready-to-use vector for rapid construction.  High levels of shRNA expression are achieved using the built-in H1 promoter.  Positively expressing cells can be sorted using the CMV promoter-driven GFP reporter (Cat# LF523A-1), RFP reporter (Cat# LF526A-1) or selected for Puromycin resistance (Cat# LF524A-1). Alternatively, you can choose to have the RFP marker expressed from an EF1 promoter (Cat# LF527A-1). See plasmid vector maps below for additional details.

One-step annealing of cost-effective, short synthetic oligos for shRNA cloning

Clone-it shRNA Vector Maps

How the shRNAs are expressed

Details of the Lentiviral Delivery System

MicroRNA Precursor Cloning System Details

Description	Vector Maps
MicroRNA precursor genes are cloned into the 3' end of the copGFP or RFP transcript where they can be processed naturally into precursor and mature microRNAs. Positively expressing cells can be sorted using a GFP or RFP reporter.

Cloning Efficiency Data

 

Sample MicroRNA Expression Data

miR-10b construct cloned using Clone-it microRNA Cloning System was transiently transfected into 293 cells with the indicated amount of plasmid DNA.  Mature miR-10b was measured using QuantiMir™.

Details of the Lentiviral Delivery System

Transcription Reporter Cloning System Details

The easiest method for efficient & accurate cloning of Transcription Response Elements or Promoter sequences with ready-to-use (pre-linearized) Clone-it vectors that require no restriction digestion.

Features and Benefits of the Clone-it™ Cloning System Ligase, recombinase, topoisomerase and restriction enzyme-free system Rapid and Directional cloning with no recombination errors Near 100% cloning efficiency Short hands-on time and adaptable for high-throughput cloning Lentiviral-based expression systems containing HIV backbone for high viral titers Reliable delivery to dividing or non-dividing cells Convenient sorting of transfected or transduced cells Easily create stable cell lines

 

Transcription Reporter Cloning System Details

Transcription Response Element or Promoter sequences are cloned downstream of an inactive, minimal CMV ("mCMV") promoter.  Transcriptional activity driven by your promoter sequences generate a robust fluorescent marker response.  Positively expressing cells can be sorted using a RFP reporter (Cat# LF528A-1).  Details for enzyme-free cloning can be found in the User Manual.

Clone-it Reporter Vector Map Download Vector Sequence: LF528A-1• LF529A-1

Sample Transcription Reporter Data Murine neural stem cells reporting BDNF promoter transactivation four weeks after differentiation. Visualization of single cells (left) and neurosphere (right).

Details of the Lentiviral Delivery System

Online Resources

 

Product Citations

Nassirpour R, Shao L, Flanagan P, Abrams T, Jallal B, Smeal T, Yin MJ. Nek6 mediates human cancer cell transformation and is a potential cancer therapeutic target. Mol Cancer Res. 2010 May;8(5):717-28.

Technical References

de Jong, RN et al. Enzyme Free Cloning for high throughput gene cloning and expression. (2006) J. Struct Funct Genomics. 7: 109-118.

Tillett, D. and Neilan, BA. Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites. (1999) Nucleic Acids Research 27:e26-e26.

Viral vectors for gene therapy. Methods and Protocols. Eds. C.A. Machida. (2003), Humana Press.

Methods in Molecular Biology. Volume 246. Gene delivery to mammalian cells. Volume 2: Viral Gene transfer techniques. Ed. by W. C. Heiser. (2004), Humana Press.

Methods in Molecular Biology. Volume 229. Lentivirus gene engineering protocols. Ed. by M. Federico. (2003), Humana Press.

Li MJ, Rossi JJ. Lentiviral vector delivery of recombinant small interfering RNA expression cassettes. Methods Enzymol. 2005;392:218-26.

Davidson BL, Harper SQ. Viral delivery of recombinant short hairpin RNAs.
Methods Enzymol. 2005;392:145-73.

Clone-it Lentivector Systems Quantity Product Size Catalog # Price Ligase-Free cDNA Cloning and Expression (pPS-EF1-LCS-T2A-RFP) 20 reactions LF520A-1 $388.00 Ligase-Free cDNA Cloning and Expression (pPS-EF1-LCS-T2A-GFP) 20 reactions LF520B-1 $388.00 Ligase-Free cDNA Cloning and Expression (pPS-EF1-LCS-T2A-Puro) 20 reactions LF521A-1 $388.00 Ligase-Free miRNA Cloning and Expression (pPS-EF1-GFP-LCS) 20 reactions LF522A-1 $388.00 Ligase-Free miRNA Cloning and Expression (pPS-EF1-RFP-LCS) 20 reactions LF522B-1 $388.00 Ligase-Free shRNA Cloning and Expression (pPS-H1-LCS [GFP]) 20 reactions LF523A-1 $388.00 Ligase-Free shRNA Cloning and Expression (pPS-H1-LCS [Puro]) 20 reactions LF524A-1 $388.00 Ligase-Free shRNA Cloning and Expression (pPS-CMV-aRFP-H1-LCS [RFP]) 20 reactions LF526A-1 $388.00 Ligase-Free shRNA Cloning and Expression (pPS-EF1-aRFP-H1-LCS [RFP]) 20 reactions LF527A-1 $388.00 Ligase-Free Promoter Cloning Reporter (pPS-LCS-mCMV-RFP) 20 reactions LF528A-1 $388.00 Ligase-Free Promoter Cloning Reporter (pPS-LCS-mCMV-GFP) 20 reactions LF529A-1 $388.00 Kit Components Each kit contains sufficient vector for 20 cloning reactions. Each kit contains positive control primers and template. cDNA and microRNA kits also contain DpnI to remove any parental plasmid template.

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