pBBR1MCS3-mCherry-Tet vector (Cat. No.: V037588)
- Name:
- pBBR1MCS3-mCherry-Tet
- Antibiotic Resistance:
- Tetracycline
- Length:
- 5873 bp
- Type:
- Protein expression
- Replication origin:
- pBBR1 oriV
- Host:
- Broad host
- Selection Marker:
- mCherry
- Copy Number:
- Extremely low
- Promoter:
- T7
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
- Expression Method:
- Transient
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.
pBBR1MCS3-mCherry-Tet vector (Cat. No.: V037588) Sequence
LOCUS V037588 5873 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V037588
VERSION V037588
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
promoter 281..309
/note="E. coli promoter for tetracycline efflux protein
gene"
/label="tet promoter"
CDS 357..1547
/codon_start=1
/gene="tet"
/note="confers resistance to tetracycline"
/product="tetracycline efflux protein"
/transl_table=1
/translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST*"
/label="TcR"
protein_bind 1741..1762
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
promoter 1777..1807
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 1815..1831
/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 1839..1855
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
promoter 1876..1894
/note="promoter for bacteriophage T3 RNA polymerase"
/label="T3 promoter"
CDS 1913..2623
/codon_start=1
/note="mammalian codon-optimized"
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/transl_table=1
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK*"
/label="mCherry"
promoter complement(2668..2686)
/note="promoter for bacteriophage T7 RNA polymerase"
/label="T7 promoter"
primer_bind complement(2696..2712)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 fwd"
CDS complement(3419..4081)
/codon_start=1
/experiment=""
/gene=""
/note=""
/product="replication protein for the broad-host-range
plasmid pBBR1 from Bordetella bronchiseptica"
/protein_id=""
/transl_table=1
/translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR*"
/label="pBBR1 Rep"
rep_origin 4082..4851
/note="replication origin of the broad-host-range plasmid
pBBR1 from Bordetella bronchiseptica; requires the pBBR1
Rep protein for replication"
/label="pBBR1 oriV"