Basic Vector Information
- Vector Name:
- pLX304-Flag-APEX2-NES
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8553 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Selection Marker:
- Blasticidin
- Promoter:
- CMV
- Cloning Method:
- Gateway Cloning
- 5' Primer:
- CMV-F
- 3' Primer:
- WPRE-R
pLX304-Flag-APEX2-NES vector Vector Map
Plasmid Resuspension 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 ℃.
pLX304-Flag-APEX2-NES vector Sequence
LOCUS 40924_29471 8553 bp DNA circular SYN 13-MAY-2021
DEFINITION Cytosolic APEX2 construct.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8553)
AUTHORS Hung V, Lam SS, Udeshi ND, Svinkina T, Guzman G, Mootha VK, Carr SA,
Ting AY
TITLE Proteomic mapping of cytosol-facing outer mitochondrial and ER
membranes in living human cells by proximity biotinylation.
JOURNAL Elife. 2017 Apr 25;6. pii: e24463. doi: 10.7554/eLife.24463.
PUBMED 28441135
REFERENCE 2 (bases 1 to 8553)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8553)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Elife.";
date: "2017-04-25"; pages: "
10.7554/eLife.24463"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8553
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin complement(154..742)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(916..1773)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(1774..1878)
/label=AmpR promoter
rep_origin complement(1904..2359)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 2501..2517
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 2527..2545
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin complement(2566..2701)
/direction=LEFT
/label=SV40 ori
/note="SV40 origin of replication"
polyA_signal complement(2728..2862)
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
LTR complement(2940..3173)
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
misc_feature complement(3245..3833)
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(3875..3916)
/codon_start=1
/label=V5 tag
/note="epitope tag from simian virus 5"
/translation="GKPIPNPLLGLDST"
protein_bind 3918..3942
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
CDS complement(3950..3976)
/codon_start=1
/label=NES
/note="nuclear export signal from the HIV Rev protein
(Fischer et al., 1995)"
/translation="LPPLERLTL"
CDS complement(3983..4729)
/codon_start=1
/label=APEX2
/note="soybean ascorbate peroxidase, engineered to serve as
a monomeric tag for electron microscopy and live-cell
proteomics (Lam et al., 2015)"
/translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
YAADEDAFFADYAEAHQKLSELGFADA"
CDS complement(4730..4753)
/codon_start=1
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/translation="DYKDDDDK"
protein_bind complement(4774..4798)
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
promoter complement(4821..5024)
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
enhancer complement(5025..5328)
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
misc_feature complement(5445..5562)
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
CDS complement(5622..6017)
/codon_start=1
/label=BSD
/note="blasticidin S deaminase"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
promoter complement(6029..6529)
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS complement(6704..6748)
/codon_start=1
/label=gp41 peptide
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
/translation="KNEQELLELDKWASL"
misc_feature complement(6933..7166)
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature complement(7659..7784)
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
LTR 7831..8011
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
promoter complement(8012..8238)
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
promoter complement(8266..8284)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(8305..8321)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(8329..8345)
/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(8353..8383)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(8398..8419)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(8536..8553)
/label=L4440
/note="L4440 vector, forward primer"
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