pCE-mp53DD vector (V006892)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V006892	pCE-mp53DD	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:: pCE-mp53DD

Antibiotic Resistance:: Ampicillin

Length:: 9690 bp

Type:: Mammalian Expression

Replication origin:: ori

Copy Number:: High Copy

Promoter:: CAG

Cloning Method:: Restriction Enzyme

5' Primer:: pCAG-F

3' Primer:: TTAGCCAGAAGTCAGATGCTC

pCE-mp53DD vector Map

Copy Sequence View Map

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.

pCE-mp53DD vector Sequence

LOCUS       40924_10326        9690 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Non-integrating (episomal) expression of mouse p53DD - p53 
            carboxy-terminal dominant-negative fragment .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9690)
  AUTHORS   Okita K, Yamakawa T, Matsumura Y, Sato Y, Amano N, Watanabe A, 
            Goshima N, Yamanaka S
  TITLE     An Efficient Non-viral Method to Generate Integration-Free Human iPS
            Cells from Cord Blood and Peripheral Blood Cells.
  JOURNAL   Stem Cells. 2012 Nov 29. doi: 10.1002/stem.1293.
  PUBMED    23193063
REFERENCE   2  (bases 1 to 9690)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9690)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Stem Cells.
            2012 Nov 29. doi: 10.1002/stem.1293."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids,
                     forward primer"
     primer_bind     complement(2143..2162)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2208..2263
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2262..2281)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also
                     called rb-glob-pA-term-R"
     primer_bind     complement(2624..2640)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2648..2664)
                     /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(2672..2702)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2717..2738)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3217..5136
                     /codon_start=1
                     /product="Epstein-Barr nuclear antigen 1, also known as
                     EBNA-1"
                     /label=EBNA1
                     /note="crucial for latent viral infection, and for episomal
                     amplification of vectors containing the oriP origin (Okita 
                     et al., 2013)"
                     /translation="MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRGR
                     GRGRGGGRPGAPGGSGSGPRHRDGVRRPQKRPSCIGCKGTHGGTGAGAGAGGAGAGGAG
                     AGGGAGAGGGAGGAGGAGGAGXGGGXGAGGGAGGXGGXGXGGGAGXGGGAGGAGAGGGA
                     GGGGGXGAGGGAGAGGGAGGAGAGGAGGAGGAGGGGGAGXGGGGGAGGAGAGGAGAGGG
                     AGGAGGAGAGGAGAGGAGAGGAGGGGAGGAGAGGAGGAGAGGAGGAGAGGAGGAGAGGG
                     AGGAGAGGGGGXGXGGXGGXGAGGAGGAGAGGGAGAGGAGAGGGGRGRGGSGGRGRGGS
                     GGRGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSGSPPRRPPPG
                     RRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRGQGDGGRRK
                     KGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVFVYGGSKTSLYN
                     LRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLKDA
                     IKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDEGE
                     EGQE"
     rep_origin      5438..7227
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
     primer_bind     complement(7768..7785)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7939..8527)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8701..9558)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(9559..9663)
                     /label=AmpR promoter
     promoter        9690
                     /label=CAG
                     /note="CMV early enhancer fused to modified chicken 
                     beta-actin promoter"