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.
| 载体基本信息 | |||
| 载体名称 | pGluc-Basic | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 4920 bp | 载体类型 | Promoter enhancer reporter vector |
| 克隆方法 | Enzyme digestion and ligation | 5引物 | 5′ d(GGGGTTCCGCGCACATTTCCCCG) 3′ |
pGluc-Basic 载体载体图谱
pGluc-Basic 载体载体序列
LOCUS Exported 4920 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4920)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4920
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 76..633
/codon_start=1
/product="secreted Gaussia luciferase"
/note="hGLuc"
/note="human codon-optimized"
/translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
KLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
QGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
RCATFASKIQGQVDKIKGAGGD"
rep_origin 786..1214
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1228..1558
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 1409..1544
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 1625..2419
/codon_start=1
/gene="aph(3)-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 2593..2714
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(2763..2779)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2787..2803
/bound_moiety="lac repressor encoded by lacI"
/note="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)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 2856..2877
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3165..3753)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3924..4784)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4785..4889)
/gene="bla"
/note="AmpR promoter"
