pECFP-Mem encodes a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin, also called GAP-43 (1), and a cyan fluorescent variant of the enhanced green fluorescent protein (EGFP). The neuromodulin fragment contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets ECFP to cellular membranes. The ECFP gene contains amino acid substitutions that give ECFP fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm) and emission (major peak at 475 nm and a minor peak at 501 nm) spectra similar to other cyan emission variants (2, 3). These substitutions also enhance the brightness and solubility of the protein (2, 4, 5). In addition to the chromophore mutations, ECFP contains over 190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (6, 7). Furthermore, upstream sequences flanking the ECFP-Mem fusion protein have been converted to a Kozak consensus translation initiation site (8). These changes increase the translational efficiency of the fusion protein and consequently its expression in mammalian cells.Expression of ECFP-Mem is driven by the immediate early promoter of CMV ( P CMV IE ). The vector contains an SV40 origin of replication and a neomycin resistance (Neo r ) gene for selection in mammalian cells. A bacterial promoter upstream of this cassette ( P ) expresses kanamycin resistance in E. coli . The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.pECFP-Mem can be transfected into mammalian cells using any standard method. If required, stable transformants can be selected using G418 (9). Expression of ECFP-Mem in mammalian cells results in strong labeling of the plasma membrane and allows easy tracking of individual cells in a population. This membrane labeling also permits study of fine cellular processes such as neuronal axons (10), leading edges of migrating cells, filopodia, or microvilli on cell surfaces. pECFP-Mem cannot be used as an exclusive plasma membrane marker because it also partially labels intracellular membranes.
| 载体基本信息 | |||
| 载体名称 | pECFP-MEM | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4793 bp | 载体类型 | Fluorescent Protein Reporter Vectors |
| 筛选标记 | Neomycin | 启动子 | CMV |
| 载体标签 | ECFP | ||
pECFP-MEM 载体载体图谱
pECFP-MEM 载体载体序列
LOCUS Exported 4793 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 4793)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4793
/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"
misc_feature 591..671
/note="MCS"
/note="multiple cloning site"
CDS 739..1458
/codon_start=1
/product="enhanced CFP"
/note="ECFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 1581..1702
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1709..2164)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2191..2295
/gene="bla"
/note="AmpR promoter"
promoter 2297..2654
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2505..2640
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2689..3483
/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 3715..3762
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4091..4679
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
