pAcGFP1-C载体描述 protein (GFP) from Aequorea coerulescens. The fluorescent protein coding sequence in this construct has been human-codon-optimized for efficient expression and enhanced brightness. AcGFP1 protein has an excitation maximum at 475 nm and an emission maximum at 505 nm. Sequences flanking AcGFP1 have been converted to a Kozak consensus translation initiation site (1) to further increase translation efficiency in eukaryotic cells. A gene of interest can be added in-frame downstream of the AcGFP1 coding sequence and expressed as a fusion protein to the C-terminus of AcGFP1. SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3’end of the mRNA transcript. In addition, the vector also contains a SV40 origin of replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor) containing an SV40 early promoter, the neomycin/ kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows selection of stable transformants in eukaryotic cells using G418. A bacterial promoter upstream of the gene expresses kanamycin resistance in E. coli. pAcGFP-C Vector also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
Provided with the Matchmaker Chemiluminescent CoIP Vector Set, the pAcGFP1-C Vector allows the generation of a N-terminal AcGFP1-bait fusion. Any bait sequence in our current Matchmaker Yeast Two Hybrid System 3 pGBKT7 bait vector can be PCR-amplified using the AcGFP BD FWD/REV universal In-Fusion primer set, and directionally and efficiently cloned into the SalI/HindIII sites of the pAcGFP1-C using our In-Fusion Dry-Down PCR Cloning Kit. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard transfection method. The resulting expressed AcGFP1- bait fusion protein will automatically be in-frame and can be used to monitor transfection efficiency as well as AcGFP1-bait fusion expression and localization in vivo since the AcGFP1-bait fusion retains the fluorescent properties of the native AcGFP1 protein.
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. Kozak, M. (1987) Nucleic Acids Res. 15(20):8125–8148. 2. Gorman, C. (1985) In DNA cloning: A practical approach, Vol. II. Ed. D.M. Glover. (IRL Press, Oxford, UK) pp. 143–190.
