pEGFP-Tub encodes a fusion protein consisting of the red-shifted, human codon-optimized variant of green fluorescent protein (EGFP; 1–3) and the gene encoding human α-tubulin (4, 5). EGFP, a derivative of the GFPmut1 variant (6), has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) This variant contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr, as well as more than 190 silent base changes that correspond to human codon-usage preferences (7). SV40 polyadenylation signals downstream of the EGFP-Tub 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. A bacterial promoter upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli. The pEGFP-Tub backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.The pEGFP-Tub Vector is designed for expressing the EGFP-tubulin fusion protein in mammalian cells. The protein incorporates directly into microtubules and thereby allows the observation of microtubules in living or fixed cells by fluorescence microscopy (5, 8). pEGFP-Tub can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (9).
| 载体基本信息 | |||
| 载体名称 | pEGFP-Tub | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 6045 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 | EGFP |
pEGFP-Tub 载体载体图谱
pEGFP-Tub 载体载体序列
LOCUS Exported 6045 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 6045)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..6045
/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 GFP"
/note="EGFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 2833..2954
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(2961..3416)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3443..3547
/gene="bla"
/note="AmpR promoter"
promoter 3549..3906
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 3757..3892
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 3941..4735
/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 4967..5014
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 5343..5931
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
