Mouse G6pc1 ORF clone (NM_008061) (Cat. No.: V046434)
- Name:
- pT3-EF1a-G6pc1(mouse)-FLAG
- Accession ID:
- NM_008061.4
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6536 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells
- Copy Number:
- High
- Promoter:
- EF1a
- Fusion Tag:
- FLAG
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
- Expression Method:
- Transient
- Gene Synonyms:
- G6Pase; G6pc; G6pt; Glc-6-Pase
- Transcript Definition:
- Mus musculus glucose-6-phosphatase catalytic subunit 1 (G6pc1), 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.
Mouse G6pc1 ORF clone (NM_008061) (Cat. No.: V046434) Sequence
LOCUS V046434 6536 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V046434
VERSION V046434
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
promoter 1..1179
/note="strong constitutive promoter for human elongation
factor EF-1α"
/label="EF-1α promoter"
promoter 1196..1214
/note="promoter for bacteriophage T7 RNA polymerase"
/label="T7 promoter"
protein_bind 1293..1317
/bound_moiety="BP Clonase™"
/gene="mutant version of attB"
/note="recombination site for the Gateway® BP reaction"
/label="attB1"
CDS 1341..1364
/codon_start=1
/product="FLAG® epitope tag, followed by an enterokinase
cleavage site"
/transl_table=1
/translation="DYKDDDDK"
/label="FLAG"
CDS 1365..2435
/label="G6pc1(NM_008061)"
/note="G6pc1(NM_008061)"
/gene="G6pc1"
polyA_signal 2464..2688
/note="bovine growth hormone polyadenylation signal"
/label="bGH poly(A) signal"
protein_bind 2758..2791
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
/label="loxP"
primer_bind complement(3249..3265)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 fwd"
promoter 3739..3843
/gene="bla"
/label="AmpR promoter"
CDS 3844..4704
/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 4875..5463
/direction=RIGHT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
protein_bind 5751..5772
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
promoter 5787..5817
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 5825..5841
/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 5849..5865
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
protein_bind 6363..6396
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
/label="loxP"