Human NMNAT1 ORF clone (NM_001297778) (Cat. No.: V018788)
- Name:
- pAAV-NMNAT1(human)-FLAG
- Accession ID:
- NM_001297778.1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5502 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells, Adeno-associated virus
- Promoter:
- CMV
- Fusion Tag:
- FLAG
- Expression Method:
- Transient
- Gene Synonyms:
- LCA9; NMNAT; PNAT1; SHILCA
- Transcript Definition:
- Homo sapiens nicotinamide nucleotide adenylyltransferase 1 (NMNAT1), transcript variant 2, 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 NMNAT1 ORF clone (NM_001297778) (Cat. No.: V018788) Sequence
LOCUS V018788 5502 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V018788
VERSION V018788
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
repeat_region 1..141
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
/rpt_type=inverted
/label="AAV2 ITR"
enhancer 221..524
/note="human cytomegalovirus immediate early enhancer"
/label="CMV enhancer"
promoter 525..727
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
intron 876..1254
/note="fusion between human cytomegalovirus intron A and
human β-globin intron 2"
/label="chimeric intron"
CDS 1349..2185
/label="NMNAT1(NM_001297778)"
/note="NMNAT1(NM_001297778)"
/gene="NMNAT1"
CDS 2186..2209
/codon_start=1
/product="FLAG® epitope tag, followed by an enterokinase
cleavage site"
/transl_table=1
/translation="DYKDDDDK"
/label="FLAG"
polyA_signal 2249..2725
/note="human growth hormone polyadenylation signal"
/label="hGH poly(A) signal"
repeat_region 2765..2905
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
/rpt_type=inverted
/label="AAV2 ITR"
rep_origin 2980..3435
/direction=RIGHT
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
/label="f1 ori"
promoter 3717..3821
/gene="bla"
/label="AmpR promoter"
CDS 3822..4682
/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 4853..5441
/direction=RIGHT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"