Basic Vector Information
- Vector Name:
- pMK284 (Stag-3FLAG-Hygro)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5589 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- Mammalian cells
- Selection Marker:
- Hyg
- Promoter:
- mPGK
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
pMK284 (Stag-3FLAG-Hygro) 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 ℃.
pMK284 (Stag-3FLAG-Hygro) vector Sequence
LOCUS 62056_17030 5589 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5589)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5589
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 4..459
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 600..616
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 626..644
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 677..721
/codon_start=1
/label=S-Tag
/note="affinity and epitope tag derived from pancreatic
ribonuclease A"
/translation="KETAAAKFERQHMDS"
CDS 734..799
/codon_start=1
/label=3xFLAG
/note="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
polyA_signal 931..1052
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
protein_bind 1083..1116
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
promoter 1177..1676
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 1737..2753
/codon_start=1
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
/translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
polyA_signal 2839..3292
/label=PGK poly(A) signal
/note="mouse phosphoglycerate kinase 1 polyadenylation
signal"
promoter complement(3401..3419)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(3440..3456)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(3464..3480)
/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(3488..3518)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(3533..3554)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3842..4430)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4604..5461)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5462..5566)
/label=AmpR promoter
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