Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007376 | pTubb3-MC | 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:
- pTubb3-MC
- Antibiotic Resistance:
- Kanamycin
- Length:
- 4885 bp
- Type:
- CRISPR
- Replication origin:
- ori
- Copy Number:
- High Copy
- Cloning Method:
- Ligation Independent Cloning
pTubb3-MC vector 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.
pTubb3-MC vector Sequence
LOCUS 40924_44414 4885 bp DNA circular SYN 13-MAY-2021
DEFINITION minicircle parental backbone plasmid including GFP knock-in donor
targeting Tubb3 gene and one cutting site for HITI .
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4885)
AUTHORS Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ,
Hatanaka F, Yamamoto M, Araoka T, Li Z, Kurita M, Hishida T, Li M,
Aizawa E, Guo S, Chen S, Goebl A, Soligalla RD, Qu J, Jiang T, Fu X,
Jafari M, Esteban CR, Berggren WT, Lajara J, Nunez-Delicado E,
Guillen P, Campistol JM, Matsuzaki F, Liu GH, Magistretti P, Zhang
K, Callaway EM, Zhang K, Belmonte JC
TITLE In vivo genome editing via CRISPR/Cas9 mediated homology-independent
targeted integration.
JOURNAL Nature. 2016 Dec 1;540(7631):144-149. doi: 10.1038/nature20565. Epub
2016 Nov 16.
PUBMED 27851729
REFERENCE 2 (bases 1 to 4885)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 4885)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/nature20565"; journalName: "Nature"; date: "2016-12-1- 1";
volume: "540"; issue: "7631"; pages: "144-149"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4885
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(743..760)
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
protein_bind 781..814
/label=attB
/note="minimal attB site for the phi-C31 integrase (Groth
et
al., 2000)"
CDS 862..1575
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
CDS complement(1711..2502)
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(2866..2883)
/label=pBAD Reverse
/note="For vectors with E. coli araBAD promoter, reverse
primer"
terminator 3036..3122
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 3214..3241
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
rep_origin 3375..3963
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4117..4134
/label=L4440
/note="L4440 vector, forward primer"
misc_feature complement(4149..4289)
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 4375..4397
/label=pGEX 3'
/note="pGEX vectors, reverse primer"