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. ECFPs 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.
| 载体基本信息 | |||
| 载体名称 | pECFP-ER | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4769 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 | ||
pECFP-ER 载体载体图谱
pECFP-ER 载体载体序列
LOCUS Exported 4769 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 4769)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4769
/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 648..1361
/codon_start=1
/product="enhanced CFP"
/note="ECFP"
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
misc_feature 1377..1433
/note="MCS"
/note="multiple cloning site"
polyA_signal 1557..1678
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1685..2140)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2167..2271
/gene="bla"
/note="AmpR promoter"
promoter 2273..2630
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2481..2616
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2665..3459
/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 3691..3738
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4067..4655
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
