IP-Free Electra DAUGHTER Vectors Mammalian with CMV promoter

Transcription

1 IP-Free Electra DAUGHTER Vectors Mammalian with CMV promoter Electra cloning DNA2.0 has developed a simple one-tube universal cloning process that can be performed in a 5 minute bench-top reaction with the fidelity of a restriction based cloning system. This process leaves no cloning scars and does not require PCR or other mutation-inducing amplification. The Electra system uses the type IIS restriction enzyme SapI, which recognizes a 7bp non-palindromic recognition sequence and leaves a 3bp 5 overhang after digestion. The Electra Vector system is available for both R&D and commercial applications, and is IP-Free with no licensing restrictions A gene that is provided in a MOTHER vector can be quickly and efficiently moved into any DAUGHTER vector allowing the gene to be tested under different contexts promoters, ribosome binding sites, C- and N-terminal tags and/or fusions. DNA2.0 has constructed a large collection of IP-Free bacterial, mammalian and yeast DAUGHTER expression vectors. Any vector can be easily converted to function as an Electra vector, and DNA2.0 will assist anyone who wishes to do so. Description Mammalian with CMV promoter The IP-Free Electra DAUGHTER Mammalian Vectors are available with a CMV promoter and choice of C- and N- terminal fusions, origins of replication, resistance markers, IRES and 2A peptide for bicistronic expression that allows monitoring of your gene expression by coupled fluorescent protein expression. The Electra DAUGHTER vector is provided as a linearized vector with a 3 -TAC-5 overhang at the 5 end and a 5 -GGT-3 overhang at the 3 end. Electra DAUGHTER Vectors Mammalian with CMV promoter (10 Rx Package size) Cat # Name Feature Resistance Marker Bacterial origin pd600 CMV-ORF no fusion none puc, high copy pd602 CMV-ORF no fusion Zeocin puc, high copy pd603 CMV-ORF no fusion Neomycin puc, high copy pd607 CMV-ORF no fusion Hygromycin puc, high copy pd608 CMV-ORF no fusion Blasticidin puc, high copy pd609 CMV-ORF no fusion Puromycin puc, high copy pd610 CMV-ORF no fusion none pacyc, low copy pd612 CMV-ORF no fusion Zeocin pacyc, low copy pd613 CMV-ORF no fusion Neomycin pacyc, low copy pd617 CMV-ORF no fusion Hygromycin pacyc, low copy pd618 CMV-ORF no fusion Blasticidin pacyc, low copy pd619 CMV-ORF no fusion Puromycin pacyc, low copy pd657-ra CMV-RFP-2A-ORF RFP-2A-ORF Hygromycin puc, high copy

2 pd677-nc CMV-GFP-ORF N-term CometGFP fusion Hygromycin puc, high copy pd677-cc CMV-ORF-GFP C-term CometGFP fusion Hygromycin puc, high copy pd668-ac CMV-ORF-2A-GFP ORF-2A-GFP Blasticidin puc, high copy pd678-nc CMV-GFP-ORF N-term CometGFP fusion Blasticidin puc, high copy pd678-cc CMV-ORF-GFP C-term CometGFP fusion Blasticidin puc, high copy pd659-ca CMV-GFP-2A-ORF GFP-2A-ORF Puromycin puc, high copy pd669-ar CMV-ORF-2A-RFP ORF-2A-RFP Puromycin puc, high copy pd679-nc CMV-GFP-ORF N-term CometGFP fusion Puromycin puc, high copy pd679-nd CMV-GFP-ORF N-term DasherGFP fusion Puromycin puc, high copy pd679-cc CMV-ORF-GFP C-term CometGFP fusion Puromycin puc, high copy pd679-cd CMV-ORF-GFP C-term DasherGFP fusion Puromycin puc, high copy pd607-nls CMV-NLS-ORF Nuclear localization signal Hygromycin puc, high copy pd608-nls CMV-NLS-ORF Nuclear localization signal Blasticidin puc, high copy pd609-nls CMV-NLS-ORF Nuclear localization signal Puromycin puc, high copy pd607-caax CMV-CAAX-ORF Membrane localization Hygromycin puc, high copy pd608-caax CMV-CAAX-ORF Membrane localization Blasticidin puc, high copy pd609-caax CMV-CAAX-ORF Membrane localization Puromycin puc, high copy pd647 ORF-IRES-GFP ORF-IRES-GFP Hygromycin puc, high copy pd648 ORF-IRES-GFP ORF-IRES-GFP Blasticidin puc, high copy pd649 ORF-IRES-GFP ORF-IRES-GFP (CometGFP) Puromycin puc, high copy pd649-d ORF-IRES-GFP ORF-IRES-GFP (DasherGFP) Puromycin puc, high copy 10 Rx Package size. Vectors are linearized to leave a 5 TAC and 3 GGT overhang. Store at -20 C.

3 Vector Map (Cat #s pd600, pd602, pd603, pd607, pd608, pd609, pd617, pd618, pd619) Vector Map (Cat #s pd677-nc, pd678-nc, pd679-nc, pd679-nd, pd679-cd) DasherGFP/NLS/CAAX

4 Vector Map (Cat #s pd677-cc, pd678-cc, pd679-cc) Vector Map (Cat #s pd657-ra, pd659-ca)

5 Vector Map (Cat #s pd668-ac, pd669-ar) Vector Map (Cat #s pd647, pd648, pd649)

6 Vector images are from Gene Designer software ( When you purchase this vector, a complimentary copy of the Gene Designer file for the vector will be uploaded to your online DNA2.0 account, allowing you to view and manipulate the cloning region and all sequences. Cloning Information The DAUGHTER vectors are provided linearized with a 5 TAC and a 3 GGT overhang. Your gene in DNA2.0 s MOTHER vector or PCR product is mixed with the linearized DAUGHTER vector in the presence of Electra buffer and enzyme mix for 5 to 20 minutes at room temperature (25 C). COMPONENT VOLUME (µl) MOTHER DNA/Positive control* (20ng) 1 DAUGHTER Vector (20ng) 1 Electra Buffer Mix* 2 Electra Enzyme Mix* 1 Sterile dh 2 O 15 Total Volume 20 *Electra cloning kit reagents. The kit can be purchased separately as Cat. # EKT Combine components as listed above in single 1.5 ml tube. Incubate at room temperature for 5-20 minutes. 2. Transform 2 µl of each reaction into competent cells. 3. Plate on LB + selection antibiotic. 3a. Optionally, LB + selection antibiotic + counter- selection - streptomycin at 100 µg/ml (for selection against pmother with rpsl), Teknova Cat # L1148. OR 3b. YEG+ selection antibiotic + counter-selection - p-chloro phenylalanine at 10mM (for selection against pmother with phes), Teknova Cat # Y5700, Y Incubate plates overnight at 37 C. Pick transformants. Intellectual Property Statement Available online: