pcDNA3.1-Laconic
PVT10863 2ug
pcDNA3.1-Laconic Informaiton
Promoter: CMV, T7, SV40
Replicator: pUC ori, F1 ori, SV40 ori
Terminator: SV40 poly (A) signal, bGH poly (A) signal
Plasmid classification: Lactobacillus, lactic acid biosensor
Plasmid size: 7648bp
Prokaryotic resistance: Amp
Clonal strain: DH5 alpha
Culture conditions: 37 C, aerobic LB
Expression host: lactation cells
Induction mode: no need to induce, transient expression.
5'sequencing primers: CMV-F:CGCAAATGGGCGGTAGGCGTG
3'sequencing primers: BGH:TAGAAGGCACAGTCGAGG
pcDNA3.1-Laconic Description
Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET)-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate,
pcDNA3.1-Laconic Multiple cloning site
pcDNA3.1-Laconic Sequence
LOCUS Exported 7648 bp ds-DNA circular SYN 05-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7648)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 5, 2016 from SnapGene Viewer 3.1.4
FEATURES Location/Qualifiers
source 1..7648
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 235..614
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 863..881
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 987..1694
/codon_start=1
/product="monomeric teal (cyan) fluorescent protein (Ai et
al., 2006)"
/note="mTFP1"
/note="mammalian codon-optimized"
/translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
TNTINLEVKEGAPLPFSYDILTTAFAYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKKTTGWDASTERMYVRDGVLK
GDVKHKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYESA
VARNSTDGMDELYK"
CDS 2496..3215
/codon_start=1
/product="Venus YFP with monomerizing A206K mutation (Nagai
et al., 2002)"
/note="mVenus"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 3248..3472
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 3518..3946
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3960..4289
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 4140..4275
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 4356..5150
/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 5324..5445
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(5494..5510)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 5518..5534
/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(5542..5572)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 5587..5608
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(5896..6481)
/direction=LEFT
/note="ori"
文献和实验
技术资料