EF1a-EGFP-SKL vector (Cat. No.: V047683)
Note: This mammalian expression plasmid uses the human EF1a promoter to drive constitutive expression of an EGFP reporter protein fused to the C-terminal peroxisomal targeting signal SKL, enabling visualization of peroxisomes in live cells.
- Name:
- EF1a-EGFP-SKL
- Length:
- 5363 bp
- Type:
- Mammalian Expression Vectors
- Host:
- Mammalian cells
- Source/Author:
- Christopher Moxham Lab
- Fusion Tag:
- EGFP
- Growth Temperature:
- 37℃
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
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.
EF1a-EGFP-SKL vector (Cat. No.: V047683) Sequence
LOCUS Exported 5363 bp DNA circular SYN 25-MAR-2026
DEFINITION Mammalian Expression of Ef1a-EGFP-SKL.
ACCESSION .
VERSION .
KEYWORDS EF1a-EGFP-SKL
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5363)
TITLE Rarebase PBC plasmids
REFERENCE 2 (bases 1 to 5363)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5363
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 1..589
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 490..509
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
promoter 753..1931
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 984..1922
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 1879..1899
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
regulatory 1954..1963
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 1960..2676
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 1960..2676
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(2005..2026)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(2266..2285)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 2613..2634
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 2854..2975
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2891..2910)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 2945..2964
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(2982..3437)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3119..3140)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 3331..3350
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 3464..3568
/gene="bla"
/label=AmpR promoter
promoter 3570..3927
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
enhancer 3570..3761
/label=SV40 enhancer
/note="enhancer for the SV40 early promoter (Herr, 1993)"
primer_bind complement(3593..3613)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 3778..3913
/label=SV40 ori
/note="SV40 origin of replication"
CDS 3962..4756
/codon_start=1
/product="aminoglycoside phosphotransferase"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
CDS 3962..4756
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(4016..4035)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4626..4645
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(4944..4963)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 4988..5035
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"