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pMTL-SC7315 vector (V012834)

Price Information

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V012834 pMTL-SC7315 In stock, instant shipping

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

Basic Vector Information

Plasmid vector pMTL-SC7315, used for allele exchange in C. difficile 630. Note that pMTL-SC7215 and pMTL-SC7315 are identical except that they have the pBP1 and pCB102 Gram-positive replicons, respectively (between AscI and FseI restriction sites).

Vector Name:
pMTL-SC7315
Antibiotic Resistance:
Chloramphenicol
Length:
6000 bp
Type:
C. difficile
Replication origin:
ori
Source/Author:
Cartman ST, Kelly ML, Heeg D, Heap JT , Minton NP
Growth Strain(s):
DH5alpha
Growth Temperature:
37℃

pMTL-SC7315 vector Vector Map

pMTL-SC73156000 bp60012001800240030003600420048005400M13 revlac operatorRBScodAM13 fwdrepHChloramphenicol acetyltransferaseorioriTtraJ

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.

pMTL-SC7315 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       V012834                 6000 bp    DNA     circular SYN 04-JAN-2024
DEFINITION  Exported.
ACCESSION   V012834
VERSION     V012834
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 6000)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     63..79
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(254..278)
                     /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)."
     RBS             297..305
                     /label="Shine-Dalgarno sequence"
                     /note="full consensus sequence for ribosome-binding sites
                     upstream of start codons in E. coli; complementary to a
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             318..1598
                     /label="codA"
                     /note="E. coli cytosine deaminase"
     primer_bind     complement(1723..1739)
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     CDS             2655..2972
                     /codon_start=1
                     /gene="repH"
                     /product="putative plasmid replication protein"
                     /label="repH"
                     /protein_id="ACR43897.1"
                     /translation="MKRRAYMKTCKNCKEFIKDTEICKIHSLMIHDKTVATYCSKYNES
                     RCLHKGKVQCINCSKMNRYGWCAIKMRCFTEEEQKKERTCIKYYARSFKKAHVKKSKKK
                     K"
     gene            2655..2972
                     /gene="repH"
                     /label="repH"
     CDS             3644..4264
                     /gene="catP"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from Clostridium
                     perfringens. Accession#: P26826"
     rep_origin      4444..5032
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     oriT            5353..5462
                     /label="oriT"
                     /note="incP origin of transfer"
     CDS             5495..5863
                     /label="traJ"
                     /note="oriT-recognizing protein"