Basic Vector Information
PGK1p-Csy4-pA 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 ℃.
PGK1p-Csy4-pA vector Sequence
LOCUS 40924_21903 4199 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector PGK1p-Csy4-pA, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4199)
AUTHORS Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK.
TITLE Multiplexed and programmable regulation of gene networks with an
integrated RNA and CRISPR/Cas toolkit in human cells
JOURNAL Mol. Cell 54 (4), 698-710 (2014)
PUBMED 24837679
REFERENCE 2 (bases 1 to 4199)
AUTHORS Perli SD.
TITLE Direct Submission
JOURNAL Submitted (04-MAY-2014) Electrical Engineering and Computer Science,
Massachusetts Institute of Technology, 77, Massachusetts Avenue,
Cambridge, MA 02139, USA
REFERENCE 3 (bases 1 to 4199)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 4199)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol. Cell";
date: "2014"; volume: "54"; issue: "4"; pages: "698-710"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(04-MAY-2014) Electrical Engineering and Computer Science,
Massachusetts Institute of Technology, 77, Massachusetts Avenue,
Cambridge, MA 02139, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4199
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 72..527
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 660..1159
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 1201..1218
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
CDS 1219..1239
/codon_start=1
/label=TEV site
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
/translation="ENLYFQS"
CDS 1252..1809
/codon_start=1
/label=Csy4
/note="CRISPR-associated endoribonuclease responsible for
pre-crRNA processing in Pseudomonas aeruginosa (Haurwitz et
al., 2010)"
/translation="DHYLDIRLRPDPEFPPAQLMSVLFGKLHQALVAQGGDRIGVSFPD
LDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQA
KSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGPL
QVTAEEGGFTCYGLSKGGFVPWF"
polyA_signal complement(1985..2106)
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(2520..3108)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3282..4139)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(join(4140..4199,1..45))
/label=AmpR promoter
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