Human MBP ORF clone (NM_001025081) (Cat. No.: V042477)
- Name:
- pcDNA3.1-MBP(human)-FLAG-mCherry-SV40 NLS
- Accession ID:
- NM_001025081.2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6818 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells
- Selection Marker:
- Neo/G418;mCherry
- Copy Number:
- High
- Promoter:
- CMV
- 5' Primer:
- CMV-F
- Fusion Tag:
- FLAG-mCherry-SV40 NLS
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
- Expression Method:
- Transient
- Transcript Definition:
- Homo sapiens myelin basic protein (MBP), transcript variant 1, mRNA
Resources
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.
Human MBP ORF clone (NM_001025081) (Cat. No.: V042477) Sequence
LOCUS V042477 6818 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V042477
VERSION V042477
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
enhancer 202..581
/note="human cytomegalovirus immediate early enhancer"
/label="CMV enhancer"
promoter 582..785
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
promoter 830..848
/note="promoter for bacteriophage T7 RNA polymerase"
/label="T7 promoter"
CDS 908..1498
/label="MBP(NM_001025081)"
/note="MBP(NM_001025081)"
/gene="MBP"
CDS 1511..1534
/codon_start=1
/product="FLAG® epitope tag, followed by an enterokinase
cleavage site"
/transl_table=1
/translation="DYKDDDDK"
/label="FLAG"
CDS 1544..1600
/codon_start=1
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/product="2A peptide from porcine teschovirus-1
polyprotein"
/transl_table=1
/translation="ATNFSLLKQAGDVEENPGP"
/label="P2A"
CDS 1601..2308
/codon_start=1
/note="mammalian codon-optimized"
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/transl_table=1
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
/label="mCherry"
CDS 2309..2329
/codon_start=1
/locus_tag=""
/note=""
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/protein_id=""
/transl_table=1
/translation="PKKKRKV"
/label="SV40 NLS"
CDS 2330..2350
/codon_start=1
/locus_tag=""
/note=""
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/protein_id=""
/transl_table=1
/translation="PKKKRKV"
/label="SV40 NLS"
polyA_signal 2385..2609
/note="bovine growth hormone polyadenylation signal"
/label="bGH poly(A) signal"
rep_origin 2655..3083
/direction=RIGHT
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
/label="f1 ori"
promoter 3097..3426
/note="SV40 enhancer and early promoter"
/label="SV40 promoter"
CDS 3493..4287
/codon_start=1
/gene="aph(3')-II (or nptII)"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin®)"
/product="aminoglycoside phosphotransferase from Tn5"
/transl_table=1
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
/label="NeoR/KanR"
polyA_signal 4461..4582
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
primer_bind complement(4631..4647)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
protein_bind 4655..4671
/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-β-D-thiogalactopyranoside (IPTG)."
/label="lac operator"
promoter complement(4679..4709)
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 4724..4745
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
rep_origin complement(5033..5618)
/direction=LEFT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
CDS complement(5789..6649)
/codon_start=1
/gene="bla"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/product="β-lactamase"
/transl_table=1
/translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
SLIKHW*"
/label="AmpR"
promoter complement(6650..6754)
/gene="bla"
/label="AmpR promoter"