of efficient infection of non-proliferating cells. With the use of the third generation of packaging
system, essentially all HIV viral gene products are removed, thus eliminating the risk of producing
recombinant HIV virus. We provide cDNA subcloning service into the pLenti-CMV vector.
Expression of inserted cDNAs is powered by human cytomegalovirus immediate early gene
promoter, ensuring a high level of expression. Integration of this vector into host genome can be
selected for resistance to Blasticidin. The finished cDNA constructs can be used directly as
plasmids in transfection experiments, or packaged into virus particles for infection of proliferating
or non-proliferating cells.
To order this service, please down load the "cDNA cloning order form" (PDF, Word format) and
send your starting clone along with it to
Cellogenetics, Inc
10075 Tyler Place, Suite 6
Ijamsville, MD 21754
If you don't already have the cDNA, we also offer a service to identify and isolate a full length
clone from EST libraries. Please add this service in the order form.
If you like to mark the cDNA, you may do so in the order form. Please specify the desired tag (HA,
Myc, Flag, or other common tags) and its position (5' or 3').
We also offer to modify the cDNA for making dominant negative or constitutive active , or other
desired mutants. Please specify the desired sequence change(s) in the order form.
5'-LTR: 1-410
Packaging signal: 521-565
Rev response element RRE: 1075-1308
CMV promoter: 1809-2392
Multiple cloning site: 2401-2548
Blasticidin resistance: 3035-3500
pUC origin: 5986-6659
b-lactamase gene: 4981-5841
5' Sequencing primer: 2374-2395
5'-CTGGAGACGCCATCCACGCTG-3'
3' Sequencing primer: 2584-2563
5'-TAGGCTTACCTTCGAACCGCG-3'
Suitable host strain: DH5a, Stbl-2 and others
E. coli replication origin: pUC
Antibiotics resistance: Ampicillin (100 ug/ml)
Viral packaging cells:
Reference:
1. Dull, T et al. (1998) A third-generation of Lentiviral
vector with a conditional packaging system J. Virol.
72: 8463-8471.
2. Wang KC, Kim JA, Sivasankaran R, Segal R, He Z.
(2002) P75 interacts with the Nogo receptor as a
co-receptor for Nogo, MAG and OMgp. Nature
420: 74-78.
Purchase of the cDNA subcloning service amounts to entering a service agreement under which
Cellogenetics is contractually obligated to:
1. design and prepare DNA oligo primers,
2. perform high fidelity PCR reaction to amplify cDNA with appropriate modification,
3. subclone the PCR fragment into the pLenti-CMV vector,
4. prepare plasmid DNA and verify cDNA insert by restriction enzyme digestion,
5. sequence both ends of the finished clone to confirm correct cloning junctions and tags,
6. ship out the finished constructs as glycerol stocks.
