Human ADORA2B ORF clone (NM_000676) (Cat. No.: V021263)
- Name:
- PiggyBac-CMV-EF1a-Puro-ADORA2B(human)
- Accession ID:
- NM_000676.4
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7437 bp
- Type:
- Protein expression, Transposition
- Replication origin:
- ori
- Host:
- Mammalian cells
- Selection Marker:
- Puro
- Promoter:
- CMV
- Expression Method:
- Transient
- Gene Synonyms:
- ADORA2
- Transcript Definition:
- Homo sapiens adenosine A2b receptor (ADORA2B), 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 ADORA2B ORF clone (NM_000676) (Cat. No.: V021263) Sequence
LOCUS V021263 7437 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V021263
VERSION V021263
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
promoter complement(1..105)
/gene="bla"
/label="AmpR promoter"
rep_origin complement(131..586)
/direction=LEFT
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
/label="f1 ori"
primer_bind 728..744
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 fwd"
promoter 1442..1645
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
CDS 1734..2729
/label="ADORA2B(NM_000676)"
/note="ADORA2B(NM_000676)"
/gene="ADORA2B"
promoter 2774..2985
/note="core promoter for human elongation factor
EF-1α"
/label="EF-1α core promoter"
LTR 2998..3266
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
/label="5' LTR (truncated)"
CDS 3301..3900
/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"
polyA_signal 4008..4129
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
primer_bind complement(5416..5432)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
protein_bind 5440..5456
/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(5464..5494)
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 5509..5530
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
rep_origin complement(5818..6406)
/direction=LEFT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
CDS complement(6577..7437)
/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"