pMTL-JH4 vector (V004428)

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Price Information

Cat No.	Plasmid Name	Availability	Buy one, get one free! (?)
V004428	pMTL-JH4	In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:: pMTL-JH4

Length:: 4863 bp

Type:: Integration vector

Replication origin:: ori

Source/Author:: Heap JT, Ehsaan M, Cooksley CM, Ng YK, Cartman ST, Winzer K, Minton NP.

pMTL-JH4 vector Map

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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-JH4 vector Sequence

LOCUS       V004428                 4863 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004428
VERSION     V004428
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 4863)
  AUTHORS   Heap JT, Ehsaan M, Cooksley CM, Ng YK, Cartman ST, Winzer K, Minton
            NP.
  TITLE     Integration of DNA into bacterial chromosomes from plasmids without
            a counter-selection marker
  JOURNAL   Nucleic Acids Res. 40 (8), E59 (2012)
   PUBMED   22259038
REFERENCE   2  (bases 1 to 4863)
  AUTHORS   Heap JT, Ehsaan M, Cooksley CM, Cartman ST, Minton NP.
  TITLE     Direct Submission
  JOURNAL   Submitted (11-JAN-2011) School of Molecular Medical Sciences, The
            University of Nottingham, Centre for Biomolecular Sciences,
            University Park, Nottingham, Nottinghamshire NG7 2RD, United Kingdom
REFERENCE   3  (bases 1 to 4863)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4863)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic
            Acids Res."; date: "2012"; volume: "40"; issue: "8"; pages: "E59"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (11-JAN-2011) School of Molecular Medical Sciences, The University
            of Nottingham, Centre for Biomolecular Sciences, University Park,
            Nottingham, Nottinghamshire NG7 2RD, United Kingdom"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4863
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gene            9..822
                     /gene="pyrF fragment"
                     /label="pyrF fragment"
     misc_feature    9..306
                     /gene="pyrF fragment"
                     /note="mediates homologous recombination with the
                     chromosome of Clostridium acetobutylicum ATCC824"
     primer_bind     complement(941..957)
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     CDS             1122..1859
                     /gene="pyrK"
                     /label="Dihydroorotate dehydrogenase B (NAD(+)), electron
                     transfer subunit"
                     /note="Dihydroorotate dehydrogenase B (NAD(+)), electron
                     transfer subunit from Clostridium acetobutylicum (strain
                     ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787).
                     Accession#: Q97FS6"
     gene            1855..2304
                     /gene="pyrD fragment"
                     /label="pyrD fragment"
     CDS             2683..3123
                     /codon_start=1
                     /gene="repL"
                     /product="plasmid replication protein"
                     /label="repL"
                     /protein_id="AFA34931.1"
                     /translation="MKERYGTVYKGSQRLIDEESGEVIEVDKLYRKQTSGNFVKAYIVQ
                     LISMLDMIGGKKLKIVNYILDNVHLSNNTMIATTREIAKATGTSLQTVITTLKILEEGN
                     IIKRKTGVLMLNPELLMRGDDQKQKYLLLEFGNFEQEANEID"
     gene            2683..3123
                     /gene="repL"
                     /label="repL"
     CDS             3273..3893
                     /gene="catP"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from Clostridium
                     perfringens. Accession#: P26826"
     rep_origin      4073..4661
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"