pAcGFP1-1載體描述 pAcGFP1-1 encodes the green fluorescent protein AcGFP1, a derivative of AcGFP from Aequorea coerulescens. AcGFP1 has been optimized for brighter fluorescence. (Excitation maximum = 475 nm; emission maximum = 505 nm.) The coding sequence of the AcGFP1 gene contains silent base changes, which correspond to human codon-usage preferences (1). pAcGFP1-1 is a promoterless vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). Sequences upstream of AcGFP1 have been converted to a Kozak consensus translation initiation site (2) to enhance translation efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3' end of the AcGFP1 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists 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. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.
AcGFP1 can be used as an in vivo reporter of gene expression. Promoters should be cloned into the pAcGFP1-1 MCS upstream from the AcGFP1 coding sequence. Without addition of a functional promoter, this vector will not express AcGFP1. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard method. If required, stable transfectants can be selected using G418 (3). pAcGFP1-1載體含有以下元件： MCS: 12–83
Aequorea coerulescens green fluorescent protein (AcGFP1) gene
Kozak consensus translation initiation site: 90–100
Start codon (ATG): 97–99; Stop codon: 814-816
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 969–974 & 998–1003
mRNA 3' ends: 1007 & 1019
f1 single-strand DNA origin: 1066–1521
(Packages noncoding strand of AcGFP1-1.)
Ampicillin resistance (β-lactamase) promoter
–35 region: 1583–1588; –10 region: 1606–1611
Transcription start point: 1618
SV40 origin of replication: 1862–1997
SV40 early promoter
Enhancer (72-bp tandem repeats): 1693–1766 & 1767–1838
21-bp repeats: 1842–1862, 1863–1883 & 1885–1905
Early promoter element: 1918–1924
Major transcription start points: 1914, 1952, 1958 & 1963 Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2046–2048; stop codon: 2838–2840
G→A mutation to remove Pst I site: 2228
C→A (Arg→Ser) mutation to remove BssH II site: 2574
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3076–3081 & 3089–3094
pUC plasmid replication origin: 3425–4068
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 JM109 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
