APEX2-NLS vector (V001296)

Basic Vector Information

      • Vector Name:
      • APEX2-NLS
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 6275 bp
      • Type:
      • Mammalian Expression
      • Selection Marker:
      • V5
      • Promoter:
      • CMV
      • Cloning Method:
      • Gibson Cloning
      • 5' Primer:
      • CMV
      • 3' Primer:
      • SP6

APEX2-NLS vector Vector Map

APEX2-NLS6275 bp30060090012001500180021002400270030003300360039004200450048005100540057006000pRS-markerCMV enhancerCMV promoterT7Kozak sequenceV5 tagAPEX2SV40 NLSSV40 NLSSV40 NLSSP6 promoterBGH-revf1 oripBABE 3'NeoR/KanRSV40 poly(A) signalM13 revlac operatorlac promoterCAP binding siteL4440oriAmpRAmpR promoter

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 ℃.

APEX2-NLS vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                6275 bp ds-DNA     circular SYN 16-AUG-2021
DEFINITION  proximity biotinylation.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6275)
  AUTHORS   Kaewsapsak P, Shechner DM, Mallard W, Rinn JL, Ting AY
  TITLE     Live-cell mapping of organelle-associated RNAs via proximity 
            biotinylation combined with protein-RNA crosslinking.
  JOURNAL   Elife. 2017 Dec 14;6. doi: 10.7554/eLife.29224.
  PUBMED    29239719
REFERENCE   2  (bases 1 to 6275)
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2017-12-14"; volume: "6. doi"; pages: " 10.7554/eLife.29224"
FEATURES             Location/Qualifiers
     source          1..6275
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(44..63)
                     /note="pRS vectors, use to sequence yeast selectable 
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
     primer_bind     769..789
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             922..963
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
     CDS             964..1710
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /note="more active than the original APEX due to the A134P 
     CDS             1729..1749
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
     CDS             1753..1773
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
     CDS             1777..1797
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
     promoter        complement(1828..1846)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1829..1846)
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1866..1883)
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1872..2096
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2142..2570
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2229..2248)
                     /note="F1 origin, reverse primer"
     primer_bind     2439..2460
                     /note="F1 origin, forward primer"
     primer_bind     complement(2579..2599)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2584..2913
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2764..2899
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2826..2845
                     /note="SV40 promoter/origin, forward primer"
     CDS             2980..3774
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="confers resistance to neomycin, kanamycin, and G418 
     primer_bind     complement(3034..3053)
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3644..3663
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3948..4069
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3985..4004)
                     /note="SV40 polyA, reverse primer"
     primer_bind     4039..4058
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4118..4134)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
     primer_bind     complement(4118..4134)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4131..4153)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4142..4158
                     /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)."
     promoter        complement(4166..4196)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4211..4232
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4349..4366)
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4520..5108)
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
     primer_bind     complement(4600..4619)
                     /note="pBR322 origin, forward primer"
     CDS             complement(5279..6139)
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
     primer_bind     5902..5921
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6140..6244)
                     /label=AmpR promoter

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