Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000984 | V5-APEX2-SENP2 | 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
- Vector Name:
- V5-APEX2-SENP2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10263 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Promoter:
- hPGK
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- CMV forward
V5-APEX2-SENP2 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.
V5-APEX2-SENP2 vector Sequence
LOCUS Exported 10263 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Expression of APEX2 at nuclear pore.
ACCESSION .
VERSION .
KEYWORDS V5-APEX2-SENP2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10263)
AUTHORS Fazal FM, Han S, Parker KR, Kaewsapsak P, Xu J, Boettiger AN, Chang
HY, Ting AY
TITLE Atlas of Subcellular RNA Localization Revealed by APEX-Seq.
JOURNAL Cell. 2019 Jun 14. pii: S0092-8674(19)30555-0. doi:
10.1016/j.cell.2019.05.027.
PUBMED 31230715
REFERENCE 2 (bases 1 to 10263)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2019
Jun 14. pii: S0092-8674(19)30555-0. doi:
10.1016/j.cell.2019.05.027."
FEATURES Location/Qualifiers
source 1..10263
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 118..137
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 371..388
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 505..526
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 541..571
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 579..595
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
primer_bind 584..606
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 603..619
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 603..619
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 638..658
/label=T3
/note="T3 promoter, forward primer"
promoter 640..658
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 686..912
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 913..1093
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1140..1265
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1758..1991
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2176..2220
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
promoter 2395..2895
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
primer_bind 2814..2832
/label=hPGK-F
/note="Human PGK promoter, forward primer"
CDS 2907..3302
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=BSD
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
misc_feature 3362..3479
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
enhancer 3596..3899
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 3900..4103
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 4054..4074
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
protein_bind 4126..4150
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 4171..4212
/codon_start=1
/product="epitope tag from simian virus 5"
/label=V5 tag
/translation="GKPIPNPLLGLDST"
CDS 4213..4959
/codon_start=1
/product="soybean ascorbate peroxidase, engineered to serve
as a monomeric tag for electron microscopy and live-cell
proteomics (Lam et al., 2015)"
/label=APEX2
/note="more active than the original APEX due to the A134P
mutation"
/translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
YAADEDAFFADYAEAHQKLSELGFADA"
misc_feature 6801..7389
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(6854..6874)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(7272..7283)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 7461..7694
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 7772..7893
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(7809..7828)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 7863..7882
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 7933..8068
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 7995..8014
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(8088..8107)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(8089..8107)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(8117..8134)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(8117..8133)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8126..8148)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 8275..8730
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(8362..8381)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 8572..8593
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 8756..8860
/gene="bla"
/label=AmpR promoter
CDS 8861..9721
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(9079..9098)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin join(9892..10263,1..217)
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"