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pAB675 vector (V014424)

Price Information

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V014424 pAB675 In stock, 1 week for quality controls

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Vector Name:
pAB675
Antibiotic Resistance:
Kanamycin
Length:
10844 bp
Type:
Protein expression
Replication origin:
pBBR1 oriV
Host:
E. coli
Growth Temperature:
37℃

pAB675 vector Map

Copy Sequence View Map
pAB67510844 bp5001000150020002500300035004000450050005500600065007000750080008500900095001000010500CAP binding siteCmRcat promoterpBBR1 oriVpBBR1 RepT7 terminatorStrep-Tag IIfd terminatorTrans-2-enoyl-CoA reductase [NADH](R)-specific enoyl-CoA hydrataseRBSlac operatorT7 promoterNeoR/KanR

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.

pAB675 vector Sequence

LOCUS       V014424                10844 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V014424
VERSION     V014424
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 10844)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10844
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    89..110
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(246..878)
                     /label="CmR"
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(879..981)
                     /label="cat promoter"
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      2374..3145
                     /label="pBBR1 oriV"
                     /note="replication origin of the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1
                     Rep protein for replication"
     CDS             3146..3805
                     /label="pBBR1 Rep"
                     /note="replication protein for the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica"
     terminator      complement(4682..4729)
                     /label="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     CDS             complement(4796..4819)
                     /label="Strep-Tag II"
                     /note="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
     terminator      complement(4827..4866)
                     /label="fd terminator"
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             complement(7486..8676)
                     /gene="fabV"
                     /label="Trans-2-enoyl-CoA reductase [NADH]"
                     /note="Trans-2-enoyl-CoA reductase [NADH] from Treponema
                     denticola (strain ATCC 35405 / DSM 14222 / CIP 103919 / JCM
                     8153 / KCTC 15104). Accession#: Q73Q47"
     CDS             complement(8707..9108)
                     /gene="phaJ"
                     /label="(R)-specific enoyl-CoA hydratase"
                     /note="(R)-specific enoyl-CoA hydratase from Aeromonas
                     caviae. Accession#: O32472"
     RBS             complement(9116..9138)
                     /label="RBS"
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(9153..9177)
                     /label="lac operator"
                     /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-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(9178..9196)
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(9244..10035)
                     /label="NeoR/KanR"
                     /note="aminoglycoside phosphotransferase"