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 Vector Map
Plasmid Resuspension 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 ℃.
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"
This page is informational only.