Basic Vector Information
- Vector Name:
- pMK231 (AAVS1 CMV-MCS-PURO)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7867 bp
- Type:
- Mammalian Expression, CRISPR
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Promoter:
- hPGK
pMK231 (AAVS1 CMV-MCS-PURO) 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 ℃.
pMK231 (AAVS1 CMV-MCS-PURO) vector Sequence
LOCUS 40924_30970 7867 bp DNA circular SYN 13-MAY-2021
DEFINITION Donor for integration at the human AAVS1 locus.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7867)
AUTHORS Okumura M, Natsume T, Kanemaki MT, Kiyomitsu T
TITLE Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling
forces as a multi-arm ensemble.
JOURNAL Elife. 2018 May 31;7. pii: 36559. doi: 10.7554/eLife.36559.
PUBMED 29848445
REFERENCE 2 (bases 1 to 7867)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7867)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Elife.";
date: "2018-05-31"; pages: "
10.7554/eLife.36559"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7867
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 107..128
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 143..173
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 181..197
/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 205..221
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 242..260
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
misc_feature 295..1098
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
enhancer 1155..1458
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1459..1662
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 1685..1764
/label=MCS
/note="multiple cloning site"
polyA_signal 1884..2005
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
promoter 2006..2514
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
CDS 2536..3132
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
polyA_signal 3142..3366
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
misc_feature 3369..4205
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
promoter complement(4240..4258)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4265..4281)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
CDS 4419..4718
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
CDS 5070..5861
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
CDS complement(6117..6974)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 7098..7686
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7840..7857
/label=L4440
/note="L4440 vector, forward primer"
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