pECFP-ER vector (V006884)

Price Information

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

Basic Vector Information

pECFP-ER encodes a fusion protein consisting of enhanced cyan fluorescent protein (ECFP); the endoplasmic reticulum (ER) targeting sequence of calreticulin (1) cloned at the 5' end; and the sequence encoding the ER retrieval sequence, KDEL (2, 3), cloned at the 3' end. ECFP-ER is a soluble protein that localizes in the lumen of the ER in transfected cells. ECFP's fluorescence excitation maxima (major peak at 433 nm and a minor peak at 453 nm) and emission maxima (major peak at 475 nm and a minor peak at 501 nm) are similar to other cyan emission variants (4–6). ECFP also contains mutations to enhance the brightness and solubility of the protein, primarily due to improved protein-folding properties and efficiency of chromophore formation (5, 7, 8).In addition to the chromophore mutations, ECFP contains more than 190 silent base changes that correspond to human codon-usage preferences (9). SV40 polyadenylation signals downstream of the ECFP-ER fusion direct proper processing of the 3' end of the mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor) consisting 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 allow stably transfected eukaryotic cells to be selected using G418 (10). A bacterial promoter upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli. The pECFP-ER backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.The pECFP-ER Vector is designed for fluorescent labeling of the endoplasmic reticulum in mammalian cells (11, 12). Fluorescence can be observed in living cells by microscopy. pECFP-ER can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (10). Filter sets are available for dual-color detection of EYFP and ECFP using conventional epifluoresence microscopy (13). Please refer to the Living Colors User Manual, provided with this vector, for additional information.

pECFP-ER 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.

pECFP-ER vector Sequence

LOCUS       40924_16725        4769 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 4769)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4769)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             648..1361
                     /codon_start=1
                     /label=ECFP
                     /note="enhanced CFP"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    1377..1433
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1557..1678
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1685..2140)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2167..2271
                     /label=AmpR promoter
     promoter        2273..2630
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2665..3456
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3691..3738
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4067..4655
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"