Basic Vector Information
- Vector Name:
- pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8467 bp
- Type:
- Mammalian Expression, Retroviral, Cre/Lox
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Promoter:
- MSCV
- Cloning Method:
- Ligation Independent Cloning
- 5' Primer:
- CTCCCACAACGAGGACTACAC
pMSCV-loxp-dsRed-loxp-eGFP-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-eGFP-Puro-WPRE vector Sequence
LOCUS 40924_32345 8467 bp DNA circular SYN 13-MAY-2021
DEFINITION Conditional overexpression vector. Deletion of dsRed by Cre
recombinase results in the rapid loss of dsRed and the activation of
your gene fused to eGFP expression..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8467)
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 8467)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8467)
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..8467
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 1..517
/label=5' LTR
/note="5' long terminal repeat from murine embryonic stem
cell virus"
misc_feature 581..922
/label=MESV Psi
/note="packaging signal of murine embryonic stem cell
virus"
CDS 989..1405
/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 1439..1472
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS 1507..2181
/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(2209..2242)
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
CDS 2299..3015
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
promoter 3028..3527
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 3548..4144
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 4217..4805
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 4862..5376
/label=3' LTR
/note="3' long terminal repeat from murine embryonic stem
cell virus"
protein_bind complement(5538..5554)
/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(5562..5592)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(5607..5628)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5745..5762)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5916..6504)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6678..7535)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(7536..7640)
/label=AmpR promoter
primer_bind 7708..7726
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(7764..7786)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 7886..7905
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8099..8121
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 8249..8268
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
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