Basic Vector Information
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).
- Vector Name:
- pEGFP-Tub
- Antibiotic Resistance:
- Kanamycin
- Length:
- 6045 bp
- Type:
- Fluorescent Protein Reporter Vectors
- Replication origin:
- ori
- Selection Marker:
- Neomycin
- Promoter:
- CMV
- Cloning Method:
- Enzyme digestion and ligation
- Fusion Tag:
- EGFP
pEGFP-Tub vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
pEGFP-Tub vector Sequence
LOCUS V000717 6045 bp DNA circular SYN 13-JAN-2022
DEFINITION Exported.
ACCESSION V000717
VERSION V000717
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 6045)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 6045)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6045
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 61..364
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 613..1329
/label="EGFP"
/note="enhanced GFP"
CDS 1348..2700
/gene="Tuba1b"
/label="Tubulin alpha-1B chain"
/note="Tubulin alpha-1B chain from Rattus norvegicus.
Accession#: Q6P9V9"
polyA_signal 2833..2954
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(2961..3416)
/direction=LEFT
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3443..3547
/label="AmpR promoter"
promoter 3549..3906
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
CDS 3941..4732
/label="NeoR/KanR"
/note="aminoglycoside phosphotransferase"
polyA_signal 4967..5014
/label="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 5343..5931
/direction=RIGHT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
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