pECFP-Mito encodes a fusion of a mitochondrial targeting sequence derived from the precursor of subunit VIII of human cytochrome C oxidase (1, 2) and the enhanced cyan fluorescent protein (ECFP). The mitochondrial targeting sequence is fused to the N-terminus of ECFP. ECFP is a variant of the Aequorea victoria green fluorescent protein gene (GFP; 3–5) that 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 (6–8). The other five substitutions (Phe-64 to Leu; Ser-65 to Thr; Asn-146 to Ile; Met-153 to Thr; and Val-163 to Ala) enhance the brightness and solubility of the protein, primarily due to improved protein-folding properties and efficiency of chromophore formation (7, 9, 10). In addition to the chromophore mutations, ECFP contains >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (11). Furthermore, upstream sequences flanking ECFP have been converted to a Kozak consensus translation initiation site (12). These changes increase the translational efficiency of the ECFP-Mito mRNA and consequently the expression of ECFP-Mito in mammalian and plant cells. The vector contains an SV40 origin for replication and a neomycin resistance (Neor) gene for selection (using G418) in eukaryotic cells (13). A bacterial promoter (P) upstream of Neor expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.pECFP-Mito is designed for fluorescent labeling of mitochondria. pECFP-Mito can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (13). pECFP-Mito is not intended as a cloning vector; however, the backbone does contain unique restriction sites upstream and downstream of the ECFP-Mito sequence which permit excision of the ECFP-Mito sequence.
| 载体基本信息 | |||
| 载体名称 | pECFP-Mito | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4759 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-Mito 载体载体图谱
pECFP-Mito 载体载体序列
LOCUS Exported 4759 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 4759)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4759
/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 705..1424
/codon_start=1
/product="enhanced CFP"
/note="ECFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 1547..1668
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1675..2130)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2157..2261
/gene="bla"
/note="AmpR promoter"
promoter 2263..2620
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2471..2606
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2655..3449
/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 3681..3728
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4057..4645
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
