pECFP-Nuc encodes an enhanced cyan fluorescent variant of the Aequorea victoria green fluorescent protein (GFP) gene with three copies of the nuclear localization signal (NLS) of the simian virus 40 large T-antigen fused at its C-terminus (1, 2). The reiteration of the NLS sequence significantly increases the efficiency of translocation of ECFP into the nucleus of mammalian cells (3). The ECFP gene contains six amino acid substitutions. The Tyr-66 to Trp substitution gives 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) similar to other cyan emission variants (4-6). The five other substitutions 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 >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (9). Furthermore, upstream sequences flanking ECFP have been converted to a Kozak consensus translation initiation site (10). These changes increase the translational efficiency of the ECFP mRNA and consequently the expression of ECFP in mammalian and plant cells.The vector contains an SV40 origin for replication and a neomycin resistance (Neo r ) gene for selection (using G418) in eukaryotic cells. A bacterial promoter ( P ) upstream of Neo r 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. The ECFP-Nuc vector can be transfected into mammalian cells using any standard transfection method. If desired, stable transfectants can be selected using G418 (11).pECFP-Nuc is used for the localized expression of ECFP in the nucleus of mammalian cells. It allows the visualization of the nucleus in living and fixed cells using fluorescence microscopy. ECFP can be used for dual-labeling experiments together with EYFP or EYFP fusion proteins using standard fluorescence microscopy when the appropriate filter sets are applied. Visit here for a complete listing of recommended filter sets. pECFP-Nuc is not meant to be used as a cloning vector. However, unique restriction sites at the 5 end of ECFP, between ECFP and the three copies of the NLS, and at the 3 end of the fusion protein, allow excision or insertion of DNA.
| 载体基本信息 | |||
| 载体名称 | pECFP-Nuc | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4764 bp | 载体类型 | Fluorescent Protein Reporter Vectors |
| 筛选标记 | Neomycin | 启动子 | CMV |
| 载体标签 | ECFP | ||
pECFP-Nuc 载体载体图谱
pECFP-Nuc 载体载体序列
LOCUS Exported 4764 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 4764)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4764
/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 613..1329
/codon_start=1
/product="enhanced CFP"
/note="ECFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 1354..1374
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1378..1398
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1402..1422
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
polyA_signal 1552..1673
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1680..2135)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2162..2266
/gene="bla"
/note="AmpR promoter"
promoter 2268..2625
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2476..2611
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2660..3454
/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 3686..3733
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4062..4650
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
