Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007688 | pGluc-Basic | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pGLuc-Basic is a plasmid cloning vector capable both of replication in E. coli and stable transfection of mammalian cells. It is designed for the cloning of promoter sequences and measurement of their transcription activity using the Gaussia Luciferase Assay Kit (NEB #E3300).In E. coli, it replicates using the pMB1 origin of replication from pBR322 (although the rop gene is missing) and carries the bla (ApR) marker for selection with ampicillin. It also carries the nptII (NmR) marker under control of an SV40 promoter; thus, following transfection into mammalian cells, it can be used to form stable cell lines by selection with geneticin (G418).The multiple cloning site (MCS) is positioned immediately upstream of a promoterless reporter gene, GLuc (the humanized coding sequence for the secreted Gaussia princeps luciferase) (1), which is followed by a synthetic polyadenylation (polyA) sequence (not shown). Thus, in mammalian cells the transcriptional activity of promoter sequences cloned into the MCS can be assessed by measuring GLuc activity in the culture medium.
- Vector Name:
- pGluc-Basic
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4920 bp
- Type:
- Promoter enhancer reporter vector
- Replication origin:
- ori
- Cloning Method:
- Enzyme digestion and ligation
- 5' Primer:
- 5′ d(GGGGTTCCGCGCACATTTCCCCG) 3′
pGluc-Basic 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.
pGluc-Basic vector Sequence
LOCUS 40924_22433 4920 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4920)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 4920)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4920
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 76..630
/codon_start=1
/label=hGLuc
/note="secreted Gaussia luciferase"
/translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
KLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
QGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
RCATFASKIQGQVDKIKGAGGD"
rep_origin 786..1214
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1228..1558
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 1625..2416
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 2593..2726
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2763..2779)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(2787..2803)
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(2811..2841)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(2856..2877)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3165..3753)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3927..4784)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4785..4889)
/label=AmpR promoter