pAcGFP1-Lam載體描述 pAcGFP1-Lamin encodes a green fluorescent protein (GFP) from Aequorea coerulescens (excitation maximum = 475 nm; emission maximum = 505 nm) and the gene encoding the human nuclear lamin (1). SV40 polyadenylation signals downstream of the AcGFP1-Lamin fusion direct proper processing of the 3’end of the AcGFP1-Lamin mRNA. AcGFP1 contains silent mutations that create an open reading frame comprised almost entirely of optimized human codons. These changes increase the translational efficiency of the AcGFP1 mRNA and consequently the expression of AcGFP1 in mammalian and plant cells. The vector backbone also contains an SV40 origin for replication in any mammalian cell line that expresses the SV40 T-antigen. A neomycin resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV-TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli. The pAcGFP1-Lamin backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
The pAcGFP1-Lamin is provided in the Matchmaker Chemiluminescent Co-IP Vector Set (Cat. No. 630458) as a negative bait control construct. In mammalian cells, this vector expresses the AcGFP1-Lamin fusion protein—the AcGFP1 serves as a tag, through which a polyclonal antibody against AcGFP1 can be used for immunoprecipitation, while lamin serves as the bait for the capture of interacting proteins via coimmunoprecipitation. Because lamin does not interact with SV40 large T antigen (2), in lysates of cells cotransfected with pAcGFP1-Lamin and pProLabel-T, there is no ProLabel activity detected in the Co-IP of this control for noninteracting pairs of proteins. The fluorescence from AcGFP1-Lamin fusion protein also allows for easy detection of fusion expression and localization in vivo, as well as offering a mode for determining transfection efficiency. Propagation in E. coli Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires
a host containing an F plasmid such as JM101 or XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: ~500
Plasmid incompatibility group: pMB1/ColE1
References
1. Sturman, N. et al. (1998) J. Stuct. Biol. 122(1–2) 42–66.
2. Matchmaker Co-IP Kit (April 1999) Clontechniques XIV(2): 14–15.
3. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II. Ed. D.M. Glover. (IRL Press, Oxford, UK) pp. 143–190.
