pZsGreen1-1載體

pZsGreen1-1 encodes a human codon-optimized variant of wild-type Zoanthus sp. green fluorescent
protein, ZsGreen1 (1). The ZsGreen1 coding sequence contains a series of silent base-pair changes,
which correspond to human codon-usage preferences, for optimal expression in mammalian cells
(2). A single amino acid substitution (Asn-66 to Met) has been made to enhance the emission
characteristics of ZsGreen1 (excitation maximum = 493 nm; emission maximum = 505 nm).
pZsGreen1-1 is a promoterless ZsGreen1 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 ZsGreen1 have been converted to a Kozak consensus translation
initiation site (3) to increase translation efficiency in eukaryotic cells. SV40 polyadenylation signals
downstream of the ZsGreen1 gene direct proper processing of the 3' end of the ZsGreen1 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 singlestranded
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.


載體使用：
ZsGreen1 can be used as an in vivo reporter of gene expression. Promoters should be cloned
into the pZsGreen1-1 MCS upstream from the ZsGreen1 coding sequence. Without addition of a
functional promoter, this vector will not express ZsGreen1. The recombinant ZsGreen1 vector can
be transfected into mammalian cells using any standard transfection method. If required, stable
transfectants can be selected using G418 (4).


載體特征：
 MCS: 12–83
 Zoanthus sp. human codon-optimized Green Fluorescent Protein (ZsGreen1) gene
Kozak consensus translation initiation site: 90–100
Start codon (ATG): 97–99; Stop codon: 790–792
Asn-66 to Met mutation (A→T, C→G ): 293, 294
 SV40 early mRNA polyadenylation signal
Polyadenylation signals: 945–950 & 974–979
mRNA 3' ends: 983 & 995
 f1 single-strand DNA origin: 1042–1497
(Packages noncoding strand of ZsGreen1.)
 Ampicillin resistance (β-lactamase) promoter
–35 region: 1559–1564; –10 region: 1582–1587
Transcription start point: 1594
 SV40 origin of replication: 1838–1973
 SV40 early promoter
Enhancer (72-bp tandem repeats): 1669–1742 & 1743–1814
21-bp repeats: 1818–1837, 1839–1859 & 1861–1881
Early promoter element: 1894–1900
Major transcription start points: 1890, 1928, 1934 & 1939 Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2022–2024; stop codon: 2814–2816
G→A mutation to remove Pst I site: 2204
C→A (Arg→Ser) mutation to remove BssH II site: 2550
 Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3052–3057 & 3065–3070
 pUC plasmid replication origin: 3401–4044

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
 Plasmid incompatibility group: pMB1/Col E1


Green Fluorescent Protein (ZsGreen1)
 Excitation/Emission Maxima: 493 nm / 505 nm