Human EPHA8 ORF clone (NM_020526) (Cat. No.: V028511)
- Name:
- pLV-CMV-EPHA8(human)-Puro
- Accession ID:
- NM_020526.5
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10911 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro
- Promoter:
- CMV
- Expression Method:
- Constiutive, Stable / Transient
- Gene Synonyms:
- EEK; EK3; HEK3
- Transcript Definition:
- Homo sapiens EPH receptor A8 (EPHA8), transcript variant 1, mRNA
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
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 EPHA8 ORF clone (NM_020526) (Cat. No.: V028511) Sequence
LOCUS V028511 10911 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V028511
VERSION V028511
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
promoter 3..229
/note="Rous sarcoma virus enhancer/promoter"
/label="RSV promoter"
LTR 230..410
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
/label="5' LTR (truncated)"
misc_feature 457..582
/note="packaging signal of human immunodeficiency virus
type 1"
/label="HIV-1 Ψ"
misc_feature 1075..1308
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
/label="RRE"
CDS 1493..1537
/codon_start=1
/note="recognized by the 2H10 single-chain llama nanobody"
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/transl_table=1
/translation="KNEQELLELDKWASL"
/label="gp41 peptide"
misc_feature 1803..1920
/note="central polypurine tract and central termination
sequence of HIV-1"
/label="cPPT/CTS"
protein_bind 1938..1958
/bound_moiety="BP Clonase™"
/gene="mutant version of attB"
/note="core recombination site for the Gateway® BP
reaction"
/label="attB4"
enhancer 2019..2322
/note="human cytomegalovirus immediate early enhancer"
/label="CMV enhancer"
promoter 2323..2526
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
CDS 2566..5580
/label="EPHA8(NM_020526)"
/note="EPHA8(NM_020526)"
/gene="EPHA8"
misc_feature 5626..6214
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
/label="WPRE"
promoter 6245..6744
/note="mouse phosphoglycerate kinase 1 promoter"
/label="PGK promoter"
CDS 6761..7360
/codon_start=1
/gene="pac from Streptomyces alboniger"
/note="confers resistance to puromycin"
/product="puromycin N-acetyltransferase"
/transl_table=1
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
/label="PuroR"
LTR 7432..7665
/note="self-inactivating 3' long terminal repeat (LTR)
from HIV-1"
/label="3' LTR (ΔU3)"
polyA_signal 7737..7858
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
rep_origin 7898..8033
/note="SV40 origin of replication"
/label="SV40 ori"
promoter complement(8054..8072)
/note="promoter for bacteriophage T7 RNA polymerase"
/label="T7 promoter"
primer_bind complement(8082..8098)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 fwd"
rep_origin 8240..8695
/direction=RIGHT
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
/label="f1 ori"
promoter 8721..8825
/gene="bla"
/label="AmpR promoter"
CDS 8826..9686
/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"
rep_origin 9857..10445
/direction=RIGHT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
protein_bind 10733..10754
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
promoter 10769..10799
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 10807..10823
/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"
primer_bind 10831..10847
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
promoter 10868..10886
/note="promoter for bacteriophage T3 RNA polymerase"
/label="T3 promoter"