pDsRed2-Nuc is a mammalian expression vector that encodes Discosoma sp. red fluorescent protein (DsRed2; 1, 2) fused with three copies of the nuclear localization signal (NLS) of the simian virus 40 large T-antigen (3, 4). The NLS sequences are fused to the 3-end of DsRed2—a human codon-optimized DsRed variant engineered for faster maturation and lower non-specific aggregation (1, 5). The reiteration of the NLS sequence significantly increases the efficiency with which the DsRed2 fusion translocates into the nucleus of mammalian cells (6).To drive expression of the DsRed2 fusion, 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-NLS mRNA transcript. To further increase the translational efficiency of the DsRed2 fusion, upstream sequences have been converted to a Kozak consensus translation initiation site (7). 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 single-stranded 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—allows stably transfected eukaryotic cells to be selected using G418 (8). A bacterial promoter (P) upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli.pDsRed2-Nuc is designed for fluorescent labeling of the nucleus in living cells. The vector can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (8). The DsRed2-NLS 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 ) for more information. To detect DsRed2-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-Nuc | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4711 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-Nuc 载体载体图谱
pDsRed2-Nuc 载体载体序列
LOCUS Exported 4711 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 4711)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4711
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 59..362
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 363..566
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 611..1285
/codon_start=1
/product="improved tetrameric variant of DsRed fluorescent
protein"
/note="DsRed2"
/note="mammalian codon-optimized"
/translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
LFL"
CDS 1301..1321
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1325..1345
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1349..1369
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
polyA_signal 1499..1620
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1627..2082)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2109..2213
/gene="bla"
/note="AmpR promoter"
promoter 2215..2572
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2423..2558
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2607..3401
/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 3633..3680
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4009..4597
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
