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MXS_BidirectionalCAG vector (V000726)

Price Information

Cat No. Plasmid Name Availability Buy one, get one free! (?)
V000726 MXS_BidirectionalCAG 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:
MXS_BidirectionalCAG
Antibiotic Resistance:
Ampicillin
Length:
6221 bp
Type:
Mammalian Expression, Synthetic Biology
Replication origin:
ori
Copy Number:
High Copy
Promoter:
CAG
Cloning Method:
Restriction Enzyme
5' Primer:
TTACCGCCTTTGAGTGAG
3' Primer:
TTGTCTCATGAGCGGATAC

MXS_BidirectionalCAG vector Map

Copy Sequence View Map
MXS_BidirectionalCAG6221 bp30060090012001500180021002400270030003300360039004200450048005100540057006000chimeric intronchicken beta-actin promoterCMV enhancerCMV enhancerCMV enhancerCMV enhancerchicken beta-actin promoterchimeric intronAmpR promoterAmpRori

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.

MXS_BidirectionalCAG vector Sequence

LOCUS       40924_2169        6221 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Bidirectional CAG promoter (compatible with the MXS Chaining Kit).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6221)
  AUTHORS   Sladitschek HL, Neveu PA
  TITLE     Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors 
            in Embryonic Stem Cells.
  JOURNAL   PLoS One. 2016 May 6;11(5):e0155177. doi: 
            10.1371/journal.pone.0155177. eCollection 2016.
  PUBMED    27152616
REFERENCE   2  (bases 1 to 6221)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6221)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2016-05-6"; pages: "
            10.1371/journal.pone.0155177. eCollection 2016"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          complement(101..1117)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     promoter        complement(1118..1394)
                     /label=chicken beta-actin promoter
     enhancer        1396..1775
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     enhancer        complement(1783..2162)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     enhancer        2170..2549
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     enhancer        complement(2559..2936)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2946..3222
                     /label=chicken beta-actin promoter
     intron          3223..4239
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     promoter        4329..4433
                     /label=AmpR promoter
     CDS             4434..5291
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5465..6053
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"