RAB11A-GFP HDRT Source (pTR 143) vector (V000478)

Price Information

Cat No. Plasmid Name Availability Add to cart
V000478 RAB11A-GFP HDRT Source (pTR 143) In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
RAB11A-GFP HDRT Source (pTR 143)
Antibiotic Resistance:
Ampicillin
Length:
5814 bp
Type:
CRISPR, Synthetic Biology
Replication origin:
ori
Copy Number:
High Copy
Cloning Method:
Gibson Cloning

RAB11A-GFP HDRT Source (pTR 143) vector Map

RAB11A-GFP HDRT Source (pTR 143)5814 bp60012001800240030003600420048005400AmpRAmpR promoterM13 fwdsuperfolder GFPM13 revlac operatorlac promoterCAP binding siteori

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.

RAB11A-GFP HDRT Source (pTR 143) vector Sequence

Copy Sequence

Download GenBank File(.gb)

LOCUS       40924_48663        5814 bp DNA     circular SYN 13-MAY-2021
DEFINITION  DNA sequence source for amplifying an HDR template to tag endogenous
            human RAB11A gene with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5814)
  AUTHORS   Roth TL, Puig-Saus C, Yu R, Shifrut E, Carnevale J, Li PJ, Hiatt J, 
            Saco J, Krystofinski P, Li H, Tobin V, Nguyen DN, Lee MR, Putnam AL,
            Ferris AL, Chen JW, Schickel JN, Pellerin L, Carmody D, 
            Alkorta-Aranburu G, Del Gaudio D, Matsumoto H, Morell M, Mao Y, Cho 
            M, Quadros RM, Gurumurthy CB, Smith B, Haugwitz M, Hughes SH, 
            Weissman JS, Schumann K, Esensten JH, May AP, Ashworth A, Kupfer GM,
            Greeley SAW, Bacchetta R, Meffre E, Roncarolo MG, Romberg N, Herold 
            KC, Ribas A, Leonetti MD, Marson A
  TITLE     Reprogramming human T cell function and specificity with non-viral 
            genome targeting.
  JOURNAL   Nature. 2018 Jul 11. pii: 10.1038/s41586-018-0326-5. doi: 
            10.1038/s41586-018-0326-5.
  PUBMED    29995861
REFERENCE   2  (bases 1 to 5814)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5814)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2018 Jul 11. pii: 10.1038/s41586-018-0326-5. doi: 
            10.1038/s41586-018-0326-5."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5814
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(473..577)
                     /label=AmpR promoter
     primer_bind     1051..1067
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2309..2995
                     /codon_start=1
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Nager et al., 2011)"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGIT"
     primer_bind     complement(4265..4281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4289..4305)
                     /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(4313..4343)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4358..4379)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4667..5255)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5429..5814,1..472))
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"