Basic Vector Information
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | Clover-mRuby2-FRET-10 | Antibiotic Resistance | Kanamycin |
| Length | 5395 bp | Type | Mammalian Expression |
| Selection Marker | Neomycin (select with G418) | Copy Number | High Copy |
Clover-mRuby2-FRET-10 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 ℃.
Clover-mRuby2-FRET-10 vector Sequence
LOCUS Exported 5395 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Localization: Cytosolic FRET, Excitation: , Emission: .
ACCESSION .
VERSION .
KEYWORDS Clover-mRuby2-FRET-10
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5395)
TITLE FRET Indicator - Live and Fixed Cells
REFERENCE 2 (bases 1 to 5395)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5395
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..304
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 305..508
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 459..479
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
regulatory 535..544
/regulatory_class="other"
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 541..1251
/codon_start=1
/product="monomeric red fluorescent protein derived from
mRuby, with improved photophysical properties (Lam et al.,
2012)"
/label=mRuby2
/translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
AKFAGLGGGMDELYK"
regulatory 1276..1285
/regulatory_class="other"
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1282..2001
/codon_start=1
/product="monomeric derivative of the bright green-yellow
fluorescent protein Clover"
/label=mClover2
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(1327..1348)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind 1935..1956
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 2123..2244
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2160..2179)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 2214..2233
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(2251..2706)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2388..2409)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 2600..2619
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 2733..2837
/gene="bla"
/label=AmpR promoter
promoter 2839..3196
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(2862..2882)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 3047..3182
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3109..3128
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3231..4025
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(3285..3304)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 3895..3914
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(4213..4232)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 4257..4304
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4633..5221
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5122..5141
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
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