Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000873 | pAG426GPD-ccdB | 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:
- pAG426GPD-ccdB
- Antibiotic Resistance:
- Chloramphenicol, Ampicillin
- Length:
- 8306 bp
- Type:
- Yeast Expression ; Gateway Destination
- Replication origin:
- ori
- Host:
- Yeast
- Selection Marker:
- URA3
- Promoter:
- GPD
- Cloning Method:
- Gateway Cloning
- 5' Primer:
- GPDPro_F
pAG426GPD-ccdB 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.
pAG426GPD-ccdB vector Sequence
LOCUS 40924_4100 8306 bp DNA circular SYN 13-MAY-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8306)
TITLE S cerevisiae Advanced Gateway Destination Vectors
REFERENCE 2 (bases 1 to 8306)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8306)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8306
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(10..28)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
rep_origin 69..1411
/label=2u ori
/note="yeast 2u plasmid origin of replication"
promoter 1438..1542
/label=AmpR promoter
CDS 1543..2400
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2574..3162
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 3316..3333
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 3450..3471
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 3486..3516
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3524..3540
/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)."
primer_bind 3548..3564
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 3585..3603
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 3628..4271
/label=GAP promoter
/note="promoter for glyceraldehyde-3-phosphate
dehydrogenase; also known as the TDH3 promoter"
primer_bind 4277..4293
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 4301..4425
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
promoter 4450..4480
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 4534..5190
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 5535..5837
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(5881..6005)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
primer_bind complement(6038..6054)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
terminator 6057..6304
/label=CYC1 terminator
/note="transcription terminator for CYC1"
promoter complement(6323..6341)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(6351..6367)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 6508..6963
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(7097..7897)
/codon_start=1
/label=URA3
/note="orotidine-5'-phosphate decarboxylase, required for
uracil biosynthesis"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
promoter complement(7898..8113)
/label=URA3 promoter
primer_bind complement(8137..8156)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8256..8278
/label=pGEX 3'
/note="pGEX vectors, reverse primer"