Basic Vector Information
- Vector Name:
- pMSCV-FLIP-puro-dsRed-GFP-miRNA
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8376 bp
- Type:
- Mammalian Expression, Retroviral, RNAi, Cre/Lox
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Promoter:
- MSCV
- Cloning Method:
- Ligation Independent Cloning
- 5' Primer:
- CTCCCACAACGAGGACTACAC
pMSCV-FLIP-puro-dsRed-GFP-miRNA 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-FLIP-puro-dsRed-GFP-miRNA vector Sequence
LOCUS 40924_32325 8376 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 8376)
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 8376)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8376)
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..8376
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 341..516
/label=5' LTR (truncated)
/note="truncated long terminal repeat from Moloney murine
sarcoma virus"
misc_feature 580..921
/label=MESV Psi
/note="packaging signal of murine embryonic stem cell
virus"
CDS 988..1404
/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 complement(1417..1450)
/label=lox2272
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are
compatible with each other, but incompatible with loxP or
loxN sites (Lee and Saito, 1988)."
CDS 1467..2063
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MVEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
CDS 2094..2159
/codon_start=1
/product="2A peptide from foot-and-mouth disease virus
polyprotein"
/label=F2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="VKQTLNFDLLKLAGDVESNPGP"
CDS 2175..2849
/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(2863..2896)
/label=lox5171
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are
compatible with each other, but incompatible with loxP or
loxN sites (Lee and Saito, 1988)."
ncRNA complement(2961..3002)
/label=3' miR-155
/note="sequence downstream of the precursor of mouse
miR-155 microRNA (Uva et al., 2013)"
ncRNA complement(3066..3092)
/label=5' miR-155
/note="sequence upstream of the precursor of mouse miR-155
microRNA (Uva et al., 2013)"
CDS complement(3155..3871)
/codon_start=1
/label=EmGFP
/note="Emerald GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
protein_bind 3885..3918
/label=lox2272
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are
compatible with each other, but incompatible with loxP or
loxN sites (Lee and Saito, 1988)."
protein_bind 3965..3998
/label=lox5171
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are
compatible with each other, but incompatible with loxP or
loxN sites (Lee and Saito, 1988)."
misc_feature 4044..4632
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 4689..5204
/label=3' LTR
/note="3' long terminal repeat from murine embryonic stem
cell virus"
primer_bind complement(5373..5389)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(5397..5413)
/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(5421..5451)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(5466..5487)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5604..5621)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5775..6363)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6537..7394)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(7395..7499)
/label=AmpR promoter
primer_bind 7567..7585
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(7623..7645)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 7745..7764
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 7958..7980
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 8156..8175
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
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