Basic Vector Information
- Vector Name:
- pMSCV-loxp-dsRed-loxp-3xHA-Puro-WPRE
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7889 bp
- Type:
- Mammalian Expression, Retroviral, Cre/Lox
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Promoter:
- MSCV
pMSCV-loxp-dsRed-loxp-3xHA-Puro-WPRE 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 ℃.
pMSCV-loxp-dsRed-loxp-3xHA-Puro-WPRE vector Sequence
LOCUS 40924_32340 7889 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 7889)
AUTHORS Koo BK, Stange DE, Sato T, Karthaus W, Farin HF, Huch M, van Es JH,
Clevers H
TITLE Controlled gene expression in primary Lgr5 organoid cultures.
JOURNAL Nat Methods. 2011 Dec 4. doi: 10.1038/nmeth.1802.
PUBMED 22138822
REFERENCE 2 (bases 1 to 7889)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7889)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods. 2011 Dec 4. doi: 10.1038/nmeth.1802."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7889
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..515
/label=MSCV
/note="Murine embryonic stem cell virus promoter including
the enhancer and promoter region of PCMV virus and 5'
untranslated region of dl-587rev retrovirus"
misc_feature 579..920
/label=MESV Psi
/note="packaging signal of murine embryonic stem cell
virus"
CDS 987..1403
/codon_start=1
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
/translation="GQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTF
NVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKP
PPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
protein_bind 1437..1470
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS 1505..2179
/codon_start=1
/label=DsRed-Express2
/note="noncytotoxic tetrameric variant of DsRed fluorescent
protein (Strack et al., 2008)"
/translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
LFQ"
protein_bind complement(2207..2240)
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
CDS 2291..2317
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
CDS 2327..2353
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
CDS 2363..2389
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
promoter 2417..2916
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 2937..3533
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 3606..4194
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 4251..4765
/label=3' LTR
/note="3' long terminal repeat from murine embryonic stem
cell virus"
primer_bind complement(4934..4950)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(4958..4974)
/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)."
promoter complement(4982..5012)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(5027..5048)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5165..5182)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5336..5924)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6098..6955)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6956..7060)
/label=AmpR promoter
primer_bind 7128..7146
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(7184..7206)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 7306..7325
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 7519..7541
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 7669..7688
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
LTR 7888..7889
/label=5' LTR
/note="5' long terminal repeat from murine embryonic stem
cell virus"
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