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myc-BioID2-MCS vector (V000701)

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V000701 myc-BioID2-MCS In stock, instant shipping

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

The myc-BioID2-MCS construct is built on the pcDNA3.1 vector backbone. This tool allows for the fusion of your protein of interest to the C-terminus of BioID2, facilitating proximity-dependent biotin identification (BioID). The multiple cloning site (MCS) is positioned downstream of the myc tag and the BioID2 tag.

Vector Name:
myc-BioID2-MCS
Antibiotic Resistance:
Ampicillin
Length:
6146 bp
Type:
Mammalian Expression
Replication origin:
ori
Selection Marker:
Neomycin (select with G418)
Promoter:
CMV
Cloning Method:
Restriction Enzyme
5' Primer:
CMV-F
3' Primer:
BGH-rev
Growth Strain(s):
DH5alpha
Growth Temperature:
37℃

myc-BioID2-MCS vector Map

Copy Sequence View Map
myc-BioID2-MCS6146 bp30060090012001500180021002400270030003300360039004200450048005100540057006000pRS-markerCMV enhancerCMV promoterT7 promoterMycBioID2bGH poly(A) signalf1 oriSV40 promoterNeoR/KanRSV40 poly(A) signalM13 revlac operatorlac promoterCAP binding siteL4440oriAmpRAmpR promoter

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.

References

  • Kim, Dae In et al. “An improved smaller biotin ligase for BioID proximity labeling.” Molecular biology of the cell vol. 27,8 (2016): 1188-96. doi:10.1091/mbc.E15-12-0844

myc-BioID2-MCS vector Sequence

LOCUS       40924_2174        6146 bp DNA     circular SYN 10-JUN-2021
DEFINITION  To fuse your protein of interest to the C-terminus of BioID2 and use
            in proximity-dependent biotin identification (BioID); MCS is present
            downstream of myc tag and BioID2 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6146)
  AUTHORS   Kim DI, Jensen SC, Noble KA, Kc B, Roux KH, Motamedchaboki K, Roux 
            KJ
  TITLE     An improved smaller biotin ligase for BioID proximity labeling.
  JOURNAL   Mol Biol Cell. 2016 Feb 24. pii: mbc.E15-12-0844.
  PUBMED    26912792
REFERENCE   2  (bases 1 to 6146)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6146)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Biol 
            Cell. 2016 Feb 24. pii: mbc.E15-12-0844."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..65)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     enhancer        237..616
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        617..820
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        865..883
                     /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)"
                     /regulatory_class="other"
     CDS             910..939
                     /codon_start=1
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
                     /translation="EQKLISEEDL"
     CDS             943..1638
                     /codon_start=1
                     /label=BioID2
                     /note="promiscuous R40G mutant of a biotin protein ligase
                     from Aquifex aeolicus (Kim et al., 2016)"
                     /translation="FKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKWL
                     SQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKVS
                     GVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISEN
                     LKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKEI
                     LSGEFSLRRS"
     polyA_signal    1745..1969
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2015..2443
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2457..2786
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2853..3644
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3821..3954
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3991..4007)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4015..4031)
                     /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(4039..4069)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4084..4105)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(4222..4239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4393..4981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5155..6012)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6013..6117)
                     /label=AmpR promoter