pEGFP-Tub vector (V000717)

Price Information

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

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).

pEGFP-Tub vector Map

Plasmid 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 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

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"