Basic Vector Information
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | Cre-IRES-PuroR | Antibiotic Resistance | Ampicillin |
| Length | 9229 bp | Type | Mammalian Expression, Lentiviral |
| Selection Marker | Puromycin | Copy Number | High Copy |
| Promoter | EF1a | Cloning Method | Restriction Enzyme |
| 5' Primer | GGTTCATTCTCAAGCCTCAGACAGTG | ||
Cre-IRES-PuroR 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 ℃.
Cre-IRES-PuroR vector Sequence
LOCUS Exported 9229 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Cre-IRES-PuroR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9229)
AUTHORS Somers A, Jean JC, Sommer CA, Omari A, Ford CC, Mills JA, Ying L,
Sommer AG, Jean JM, Smith BW, Lafyatis R, Demierre MF, Weiss DJ,
French DL, Gadue P, Murphy GJ, Mostoslavsky G, Kotton DN
TITLE Generation of transgene-free lung disease-specific human induced
pluripotent stem cells using a single excisable lentiviral stem cell
cassette.
JOURNAL Stem Cells. 2010 Oct . 28(10):1728-40.
PUBMED 20715179
REFERENCE 2 (bases 1 to 9229)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Stem Cells.
2010 Oct . 28(10):1728-40."
FEATURES Location/Qualifiers
source 1..9229
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 55..74
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
LTR 266..899
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 946..1071
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1568..1801
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1986..2030
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 2323..2440
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2488..3666
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2719..3657
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 3614..3634
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
regulatory 3671..3680
/regulatory_class="other"
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3710..4738
/codon_start=1
/product="site-specific recombinase"
/label=Cre
/note="Cre is a site-specific recombinase from
bacteriophage P1. Recombination occurs at loxP sequences."
/translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
GD"
misc_feature 4742..5309
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
misc_feature 4759..5311
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
primer_bind complement(4903..4920)
/label=IRES reverse
/note="IRES internal ribosome entry site, reverse primer.
Also called pCDH-rev"
primer_bind 5130..5149
/label=IRES-F
/note="IRES internal ribosome entry site, forward primer"
CDS 5323..5922
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(5323..5342)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 5819..5839
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 5936..6524
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(5989..6009)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(6407..6418)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 6672..6852
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(6899..6917)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6985..7089
/gene="bla"
/label=AmpR promoter
CDS 7090..7950
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(7308..7327)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 8121..8709
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8610..8629
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8863..8880
/label=L4440
/note="L4440 vector, forward primer"
primer_bind 8972..8991
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
protein_bind 9120..9141
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9156..9186
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9194..9210
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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 9199..9221
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind join(9218..9229,1..5)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind join(9218..9229,1..5)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
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