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pX33 vector (V017101)

Price Information

Cat No. Plasmid Name Availability Add to cart
V017101 pX33 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:
pX33
Antibiotic Resistance:
Gentamycin
Length:
9681 bp
Type:
Gene knockout
Replication origin:
p15A ori
Host:
E. coli
Promoter:
sacB
Growth Strain(s):
DH5a
Growth Temperature:
37℃

pX33 vector Map

Copy Sequence View Map
pX339681 bp4008001200160020002400280032003600400044004800520056006000640068007200760080008400880092009600MetapyrocatechaseSK primerT7 promoterM13 fwdPc promoterGmRSacBsacB promoteroriTp15A oriCAP binding sitelac promoterlac operatorM13 revT3 promoterKS primer

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.

pX33 vector Sequence

LOCUS       V017101                 9681 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V017101
VERSION     V017101
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 9681)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             25..945
                     /gene="xylE"
                     /label="Metapyrocatechase"
                     /note="Metapyrocatechase from Pseudomonas putida.
                     Accession#: P06622"
     primer_bind     complement(3913..3929)
                     /label="SK primer"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     promoter        complement(3962..3980)
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3990..4006)
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     promoter        4545..4573
                     /label="Pc promoter"
                     /note="class 1 integron promoter"
     CDS             4762..5292
                     /label="GmR"
                     /note="gentamycin acetyltransferase"
     CDS             complement(5391..6809)
                     /label="SacB"
                     /note="secreted levansucrase that renders bacterial growth
                     sensitive to sucrose"
     promoter        complement(6810..7255)
                     /label="sacB promoter"
                     /note="sacB promoter and control region"
     oriT            7631..7740
                     /label="oriT"
                     /note="incP origin of transfer"
     rep_origin      8123..8668
                     /label="p15A ori"
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells
                     that contain a second plasmid with the ColE1 origin."
     protein_bind    9061..9082
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        9097..9127
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    9135..9151
                     /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)."
     primer_bind     9159..9175
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     promoter        9196..9214
                     /label="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     9244..9260
                     /label="KS primer"
                     /note="common sequencing primer, one of multiple similar
                     variants"