Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V004204 | 3XFlag-pA-Tn5-Fl | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- 3XFlag-pA-Tn5-Fl
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8679 bp
- Type:
- Bacterial Expression
- Replication origin:
- ori
- Promoter:
- T7
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- CGG TTT AAA CCG GGG ATC TCG
- 3' Primer:
- TTG CGC CGC AAC ATT CAC C
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
3XFlag-pA-Tn5-Fl 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.
References
- Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG, Ahmad K, Henikoff S. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun. 2019 Apr 29;10(1):1930. doi: 10.1038/s41467-019-09982-5. PMID: 31036827; PMCID: PMC6488672.
3XFlag-pA-Tn5-Fl vector Sequence
LOCUS 40924_80 8679 bp DNA circular SYN 26-MAY-2021
DEFINITION Expresses protein A and Tn5 Transposase fusion protein in bacterial
cells.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8679)
AUTHORS Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG,
Ahmad K, Henikoff S
TITLE CUT
JOURNAL Nat Commun. 2019 Apr 29;10(1):1930. doi: 10.1038/s41467-019-09982-5.
PUBMED 31036827
REFERENCE 2 (bases 1 to 8679)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8679)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Commun."; date: "2019-04-29"; pages: "
10.1038/s41467-019-09982-5"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8679
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 126..148
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(145..333)
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
protein_bind complement(856..877)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(893..1972)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(1973..2050)
/label=lacI promoter
terminator 2203..2289
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 2386..2472
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 2569..2655
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 2752..2838
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator complement(2938..2981)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
promoter 3156..3174
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 3175..3199
/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)."
RBS 3214..3236
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 3249..3314
/codon_start=1
/label=3xFLAG
/note="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 3333..3758
/codon_start=1
/label=protein A
/note="immunoglobulin binding domains of Staphylocuccus
aureus protein A"
/translation="SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHL
PNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP
NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK"
CDS 3840..5267
/codon_start=1
/label=Tn5 transposase
/note="transposase from the bacterial Tn5 transposon
(Reznikoff, 1993)"
/translation="MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKS
ITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYR
HQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESG
KWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGL
YLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNA
VLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAER
QRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTP
DECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQS
KLDGFLAAKDLMAQGIKI"
CDS 5268..5861
/codon_start=1
/label=Mxe GyrA intein
/note="modified GyrA intein (Southworth et al., 1999)"
/translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
CDS 5892..6047
/codon_start=1
/label=CBD
/note="chitin binding domain from chitinase A1 (Watanabe et
al., 1994)"
/translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
PALWQLQ"
terminator 6126..6173
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter 6233..6337
/label=AmpR promoter
CDS 6338..7195
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin complement(7240..7753)
/direction=LEFT
/label=M13 ori
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
rep_origin 7864..8452
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8606..8623
/label=L4440
/note="L4440 vector, forward primer"
primer_bind complement(8645..8664)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"