pDsRed2-Peroxi is a mammalian expression vector that encodes a fusion of Discosoma sp. red fluorescent protein (DsRed2; 1, 2) and the peroxisomal targeting signal 1 (PTS1). The PTS1 sequence is fused to the 3-end of DsRed2, a DsRed variant engineered for faster maturation and lower non-specific aggregation. The PTS1 sequence encodes the tripeptide SKL, which targets the DsRed2-PTS1 fusion protein to the matrix of peroxisomes (3–6).To drive expression of DsRed2-PTS1, this vector contains the immediate early promoter of cytomegalovirus (PCMV IE). SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3-end of the DsRed2-PTS1 mRNA transcript. Because it encodes DsRed2, a gene variant that uses human-preferred codons (7), the DsRed2-PTS1 transcript is suited for efficient translation in mammalian cells. To further increase the translational efficiency of DsRed2- PTS1, upstream sequences have been converted to a Kozak consensus translation initiation site (8). The vector also contains an SV40 origin for replication in any mammalian cell line that expresses 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—consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK poly A) gene—allow stably transfected eukaryotic cells to be selected using G418 (9). A bacterial promoter (P) upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli.pDsRed2-Peroxi is designed for fluorescent labeling of peroxisomes. The vector can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (9). The DsRed2-PTS1 fusion (excitation/emission maxima: 558 nm/ 583 nm) can be detected by fluorescence microscopy and by flow cytometry. Filter sets optimized for detecting DsRed by microscopy are available from Chroma Technology Corporation and Omega Optical Inc. Please see their websites ( www.chroma.com and www.omegafilters.com ) and the Living Colors Vol. II User Manual, provided with this vector, for more information. To detect DsRed2- PTS1-expressing cells by flow cytometry, use the instrument’s argon-ion laser to excite the fluorophore at 488 nm and the FL-2 channel to detect the fluorophore’s emission at 583 nm.
| 载体基本信息 | |||
| 载体名称 | pDsRed2-Peroxi | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4644 bp | 载体类型 | Fluorescent Protein Reporter Vectors |
| 筛选标记 | Neomycin | 启动子 | CMV |
| 克隆方法 | Enzyme digestion and ligation | ||
pDsRed2-Peroxi 载体载体图谱
pDsRed2-Peroxi 载体载体序列
LOCUS Exported 4644 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4644)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4644
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 613..1287
/codon_start=1
/product="improved tetrameric variant of DsRed fluorescent
protein"
/note="DsRed2"
/note="mammalian codon-optimized"
/translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
LFL"
polyA_signal 1432..1553
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1560..2015)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2042..2146
/gene="bla"
/note="AmpR promoter"
promoter 2148..2505
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2356..2491
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2540..3334
/codon_start=1
/gene="aph(3)-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3566..3613
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 3942..4530
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
