Basic Vector Information
ERM-APEX2 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 ℃.
ERM-APEX2 vector Sequence
LOCUS Exported 8694 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Soybean APEX2 anchored to cytosolic face of ER membrane with
N-terminal targeting sequence of residues 1-27 of ER-resident P450
oxidase 2C1..
ACCESSION .
VERSION .
KEYWORDS ERM-APEX2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8694)
AUTHORS Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK,
Ting AY
TITLE Directed evolution of APEX2 for electron microscopy and proximity
labeling.
JOURNAL Nat Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179.
PUBMED 25419960
REFERENCE 2 (bases 1 to 8694)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179."
FEATURES Location/Qualifiers
source 1..8694
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 105..408
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 409..612
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 563..583
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
protein_bind 635..659
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 836..1582
/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"
CDS 1583..1624
/codon_start=1
/product="epitope tag from simian virus 5"
/label=V5 tag
/translation="GKPIPNPLLGLDST"
protein_bind complement(1632..1656)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 1658..1699
/codon_start=1
/product="epitope tag from simian virus 5"
/label=V5 tag
/translation="GKPIPNPLLGLDST"
misc_feature 1741..2329
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(1794..1814)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(2212..2223)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 2401..2634
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 2712..2833
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2749..2768)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 2803..2822
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 2873..3008
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 2935..2954
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(3028..3047)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(3029..3047)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3057..3074)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(3057..3073)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3066..3088)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 3215..3670
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3302..3321)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3512..3533
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 3696..3800
/gene="bla"
/label=AmpR promoter
CDS 3801..4661
/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(4019..4038)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4832..5420
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5321..5340
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5574..5591
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 5708..5729
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 5744..5774
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5782..5798
/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 5787..5809
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 5806..5822
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 5806..5822
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 5841..5861
/label=T3
/note="T3 promoter, forward primer"
promoter 5843..5861
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 5889..6115
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 6116..6296
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 6343..6468
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 6961..7194
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 7379..7423
/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 7598..8098
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
primer_bind 8017..8035
/label=hPGK-F
/note="Human PGK promoter, forward primer"
CDS 8110..8505
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=BSD
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
misc_feature 8565..8682
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
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