Basic Vector Information
- Vector Name:
- MCS-13X Linker-BioID2-HA
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6335 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
MCS-13X Linker-BioID2-HA 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 ℃.
MCS-13X Linker-BioID2-HA vector Sequence
LOCUS 40924_1929 6335 bp DNA circular SYN 13-MAY-2021
DEFINITION To fuse your protein of interest to the N-terminus of BioID2 and use
in proximity-dependent biotin identification (BioID); MCS and a
flexible 25nm GS linker are present upstream of BioID2 and HA tags..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6335)
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 6335)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6335)
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..6335
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(44..63)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 235..614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 863..881
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1182..1877
/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"
CDS 1878..1904
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
polyA_signal 1932..2156
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2202..2630
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2644..2973
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 3040..3831
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 4008..4141
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4178..4194)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(4202..4218)
/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(4226..4256)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(4271..4292)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4409..4426)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4580..5168)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5342..6199)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6200..6304)
/label=AmpR promoter
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