Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006782 | Tol2-elavl3-H2B-GCaMP6s | 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:
- Tol2-elavl3-H2B-GCaMP6s
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14296 bp
- Type:
- Transposable element
- Replication origin:
- ori
- Copy Number:
- High Copy
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- ACTCCAGCAGGACCATGTGAT
- 3' Primer:
- CATCGATGGTACAGTAAAACGACGG
Tol2-elavl3-H2B-GCaMP6s 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.
Tol2-elavl3-H2B-GCaMP6s vector Sequence
LOCUS 40924_48957 14296 bp DNA circular SYN 13-MAY-2021
DEFINITION Expressed nuclear-localized GCaMP6-slow, a calcium indicator, in
neurons in zebrafish larva. The plasmid is in Tol2 transposon
backbone.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14296)
AUTHORS Freeman J, Vladimirov N, Kawashima T, Mu Y, Sofroniew NJ, Bennett
DV, Rosen J, Yang CT, Looger LL, Ahrens MB
TITLE Mapping brain activity at scale with cluster computing.
JOURNAL Nat Methods. 2014 Jul 27. doi: 10.1038/nmeth.3041.
PUBMED 25068736
REFERENCE 2 (bases 1 to 14296)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14296)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods. 2014 Jul 27. doi: 10.1038/nmeth.3041."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14296
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(144..165)
/label=F1ori-F
/note="F1 origin, forward primer"
CDS 9463..9840
/codon_start=1
/label=H2B
/note="human histone H2B"
/translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
GELAKHAVSEGTKAITKYTSAK"
CDS 9859..11208
/codon_start=1
/label=GCaMP6s
/note="improved fluorescent protein-based calcium sensor
(Chen et al., 2013)"
/translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
polyA_signal complement(11253..11387)
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(12059..12075)
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12059..12075)
/label=pBluescriptSK
/note="For pBluescript vector"
promoter complement(12112..12130)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(12151..12167)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12151..12167)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12164..12186)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 12175..12191
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(12199..12229)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(12244..12265)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12382..12399)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12553..13141)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13315..14172)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(14173..14277)
/label=AmpR promoter