Basic Vector Information
FRB-NLuc 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 ℃.
FRB-NLuc vector Sequence
LOCUS 40924_865 7145 bp DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7145) AUTHORS Luker KE, Smith MC, Luker GD, Gammon ST, Piwnica-Worms H, Piwnica-Worms D TITLE Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. JOURNAL Proc Natl Acad Sci U S A. 2004 Aug 17. 101(33):12288-93. PUBMED 15284440 REFERENCE 2 (bases 1 to 7145) TITLE Direct Submission REFERENCE 3 (bases 1 to 7145) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl Acad Sci U S A. 2004 Aug 17. 101(33):12288-93." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..7145 /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 965..1234 /codon_start=1 /label=FRB /note="FKBP-rapamycin binding domain of human FRAP" /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ" primer_bind complement(1343..1361) /label=LucNrev /note="Firefly luciferase, reverse primer" CDS 2642..2683 /codon_start=1 /label=V5 tag /note="epitope tag from simian virus 5" /translation="GKPIPNPLLGLDST" CDS 2693..2710 /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" polyA_signal 2739..2963 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3009..3437 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 3451..3781 /label=SV40 promoter /note="SV40 enhancer and early promoter" CDS 3848..4639 /codon_start=1 /label=NeoR/KanR /note="aminoglycoside phosphotransferase" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" polyA_signal 4818..4951 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4988..5004) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(5012..5028) /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(5036..5066) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(5081..5102) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5219..5236) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5390..5978) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6152..7009) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(7010..7114) /label=AmpR promoter
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