pHel3 vector (V005570)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V005570	pHel3	In stock, 1 week for quality controls	Buy one, get one free!

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

Basic Vector Information

Vector Name:: pHel3

Antibiotic Resistance:: Kanamycin

Length:: 5510 bp

Type:: Shuttle vector

Replication origin:: ori

Source/Author:: Haas R, Heuermann D.

pHel3 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.

pHel3 vector Sequence

LOCUS       40924_24443        5510 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle vector pHel3, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5510)
  AUTHORS   Haas R, Heuermann D.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-OCT-2017) Genetics and Genomics, Icahn School of 
            Medicine at Mount Sinai, 1425 Madison Ave, New York, NY 10029, USA
REFERENCE   2  (bases 1 to 5510)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5510)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (17-OCT-2017) Genetics and Genomics, Icahn School of Medicine at 
            Mount Sinai, 1425 Madison Ave, New York, NY 10029, USA"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: HGAP v. 3
            Coverage              :: 100x
            Sequencing Technology :: PacBio
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    21..76
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     rep_origin      complement(281..869)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     oriT            1095..1204
                     /label=oriT
                     /note="incP origin of transfer"
     CDS             2082..3701
                     /codon_start=1
                     /product="RepA"
                     /label=RepA
                     /protein_id="ATY70547.1"
                     /translation="MPMNTNFEQLRKQELELRKLLEELETLPQTPQIKLQKQKIQTYID
                     KITPSILSGFDQKFKEIIENLSNEFEKEKSTPLKEPQTTPTPCKDLVVSTPKDNTYTTY
                     HNNANKVNLGKLSEREANLLFAIFQKLKDQGNTLIRFEPQDLKRMIMVKSNLTNRQLLQ
                     ILKNLLDNIGGANFWIIREHVENGEIYEDHTSYMLFKQFDIRIHKPTQTIEYLEVQLND
                     SYHYLLNNLGMGGQYTSFKLLEFQRVRGKYAKTLYRLLKQYKSTGILSVEWDQFRELLD
                     IPKDYKMENIDQKVLTPSLKELRKIYPFEHLSYKKERKSHDKRKVTHIDFYFEQLPQGE
                     TKKQKQADKQRAQRDIKLIAWDIHNQIAKRNAKATIEARFLELKTLIGYQFRHNNGTIL
                     QIDNATFEKNQMFLHVSTSKNSQKFLVSNKTFALELLFVNGYSLKKDMLEIDPPSIHPI
                     TNEPIKEFAEYIGKTIHITNFNVDQCPEGINNYLKITRIVKLNDNRICVSVQDVDKPEK
                     LLKPFIAKDEKHLKNWFKKHYR"
     CDS             complement(4230..5021)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"