ptfLC3 vector (V006795)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V006795	ptfLC3	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:: ptfLC3

Antibiotic Resistance:: Kanamycin

Length:: 6136 bp

Type:: Mammalian Expression

Replication origin:: ori

Selection Marker:: Neomycin (select with G418)

Promoter:: CMV

Cloning Method:: Restriction Enzyme

ptfLC3 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.

ptfLC3 vector Sequence

LOCUS       ptfLC3.        6136 bp DNA     circular SYN 10-JAN-2024
DEFINITION  Mammalian expression of rat LC3 fused to mRFP and EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    ptfLC3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6136)
  AUTHORS   Kimura S, Noda T, Yoshimori T
  TITLE     Dissection of the autophagosome maturation process by a novel 
            reporter protein, tandem fluorescent-tagged LC3.
  JOURNAL   Autophagy.   . 3(5):452-60.
  PUBMED    17534139
REFERENCE   2  (bases 1 to 6136)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6136)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Autophagy. 
             . 3(5):452-60."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        108..411
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        412..615
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             651..1325
                     /label=mRFP1
                     /note="monomeric derivative of DsRed (Campbell et al.,
                     2002)"
     CDS             1344..2060
                     /label=EGFP
                     /note="enhanced GFP"
     CDS             2079..2501
                     /codon_start=1
                     /gene="Map1lc3b"
                     /product="rat LC3 protein"
                     /label=LC3
                     /note="Atg8 family member found on autophagosomes"
                     /translation="PSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPV
                     LDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESERDE
                     DGFLYMVYASQETFGTALAVTYMSALKATATGREPCL"
     polyA_signal    2971..3092
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(3099..3554)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3581..3685
                     /label=AmpR promoter
     promoter        3687..4044
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             4079..4870
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     primer_bind     complement(5061..5080)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5105..5152
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5481..6069
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"