pECFP-Peroxi encodes a fusion of the enhanced cyan fluorescent variant of the Aequorea victoria green fluorescent protein gene (GFP) and the peroximal targeting signal 1 (PTS1). The PTS1 sequence is fused to the 3 end of ECFP and encodes the tripeptide SKL, which targets the fusion protein to the matrix of peroxisomes (1–4). 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 (5, 6). The other five 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, 10). Furthermore, upstream sequences flanking ECFP have been converted to a Kozak consensus translation initiation site (11). These changes increase the translational efficiency of the ECFP-PTS1 mRNA and consequently the expression of ECFP-PTS1 in mammalian and plant cells.The expression of ECFP-PTS1 is driven by the immediate early promoter of CMV (PCMV IE). pECFPPeroxi contains an MCS at the 5 end of the ECFP-PTS1 sequence. Genes of interest cloned into the MCS will be expressed as fusions to the N-terminus of ECFP-PTS1 if they are in the same reading frame as ECFP-PTS1 and there are no intervening stop codons.The vector contains an SV40 origin of replication and a neomycin resistance (Neor) gene for selection (using G418) 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.Expression of ECFP-PTS1 can be used simply for the labeling of peroxisomes. The MCS at the 5 end of ECFP-PTS1 may be used to fuse genes of interest to ECFP-PTS1. The inserted gene should include the initiation ATG codon and be in frame with ECFP-PTS1. The recombinant ECFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (12). ECFP can be used for double-or triple-labeling experiments together with EYFP and DsRed using standard fluorescence microscopy and the appropriate filter sets.
| 载体基本信息 | |||
| 载体名称 | pECFP-Peroxi | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4740 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 | 载体标签 | ECFP |
pECFP-Peroxi 载体载体图谱
pECFP-Peroxi 载体载体序列
LOCUS Exported 4740 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 4740)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4740
/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 679..1395
/codon_start=1
/product="enhanced CFP"
/note="ECFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 1528..1649
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1656..2111)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2138..2242
/gene="bla"
/note="AmpR promoter"
promoter 2244..2601
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2452..2587
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2636..3430
/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 3662..3709
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4038..4626
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
