pDsRed2-Nuc vector (V001249)

Price Information

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

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.

pDsRed2-Nuc vector Map

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

pDsRed2-Nuc vector Sequence

LOCUS       40924_15710        4711 bp DNA     circular SYN 13-JAN-2022
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4711)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4711)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        59..362
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        363..566
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             611..1285
                     /codon_start=1
                     /label=DsRed2
                     /note="improved tetrameric variant of DsRed fluorescent
                     protein"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     CDS             1301..1321
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             1325..1345
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             1349..1369
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     polyA_signal    1499..1620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1627..2082)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2109..2213
                     /label=AmpR promoter
     promoter        2215..2572
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2607..3398
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3633..3680
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4009..4597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        4711
                     /label=CMV
                     /note="Human cytomegalovirus immediate early
                     enhancer/promoter"