pGluc-Basic vector (V007688) Gene synthesis in pGluc-Basic backbone

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Price Information

Cat No.	Plasmid Name	Availability	Buy one, get one free! (?)
V007688	pGluc-Basic	In stock, instant shipping

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′

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

pGluc-Basic vector Map

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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

Zeng J, Mi R, Wang Y, Li Y, Lin L, Yao B, Song L, van Die I, Chapman AB, Cummings RD, Jin P, Ju T. Promoters of Human Cosmc and T-synthase Genes Are Similar in Structure, Yet Different in Epigenetic Regulation. J Biol Chem. 2015 Jul 31;290(31):19018-33. doi: 10.1074/jbc.M115.654244. Epub 2015 Jun 10. PMID: 26063800; PMCID: PMC4521027.

Myung CH, Lee JE, Jo CS, Park JI, Hwang JS. Regulation of Melanophilin (Mlph) gene expression by the glucocorticoid receptor (GR). Sci Rep. 2021 Aug 19;11(1):16813. doi: 10.1038/s41598-021-96276-w. PMID: 34413386; PMCID: PMC8376885.

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