pEGFP-N1 vector (V012021)

Price Information

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

Basic Vector Information

The pEGFP-N1 is a vector for fusing EGFP to the C-terminus of a partner protein. It has multiple cloning sites before the GFP gene.

pEGFP-N1 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.

References

• Payán-Bravo L, Fontalva S, Peñate X, Cases I, Guerrero-Martínez JA, Pareja-Sánchez Y, Odriozola-Gil Y, Lara E, Jimeno-González S, Suñé C, Muñoz-Centeno MC, Reyes JC, Chávez S. Human prefoldin modulates co-transcriptional pre-mRNA splicing. Nucleic Acids Res. 2021 Jun 21;49(11):6267-6280. doi: 10.1093/nar/gkab446
• Medlej A, Mohammad Soltani B, Javad Mowla S, Hosseini S, Baharvand H. A novel miRNA located in the GATA4 gene regulates the expression of IGF-1R and AKT1/2 genes and controls cell proliferation. J Cell Biochem. 2020;121(5-6):3438-3450. doi:10.1002/jcb.29617
• Asselin L, Rivera Alvarez J, Heide S, et al. Mutations in the KIF21B kinesin gene cause neurodevelopmental disorders through imbalanced canonical motor activity. Nat Commun. 2020;11(1):2441. doi:10.1038/s41467-020-16294-6
• Kim HJ, Kim HJ, Kim MK, et al. SPSB1 enhances ovarian cancer cell survival by destabilizing p21. Biochem Biophys Res Commun. 2019;510(3):364-369 https://doi.org/10.1016/j.bbrc.2019.01.088
• Namyanja, Monica & Xu, Zhi-Shen & Mugasa, Claire & Lun, Zhao-Rong & Matovu, Enock & Chen, Zhengjun & Lubega, George. (2019). Preliminary evaluation of a Trypanosoma brucei FG-GAP repeat containing protein of mitochondrial localization. AAS Open Research. 2. 165. 10.12688/aasopenres.12986.1.  https://aasopenresearch.org/articles/2-165
• Abildgaard AB, Stein A, Nielsen SV, et al. Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome. Elife. 2019 https://doi.org/10.7554/eLife.49138

pEGFP-N1 vector Sequence

LOCUS       Exported                4733 bp DNA     circular SYN 26-AUG-2024
DEFINITION  Vector for fusing EGFP to the C-terminus of a partner protein. For 
            other reading frames, use pEGFP-N2 or pEGFP-N3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4733)
  AUTHORS   Clontech
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4733)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4733)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4733
                     /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"
     misc_feature    591..671
                     /label=MCS
                     /note="multiple cloning site"
     CDS             679..1395
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    1521..1642
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1649..2104)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2131..2235
                     /label=AmpR promoter
     promoter        2237..2594
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2629..3420
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3655..3702
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4031..4619
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"