Search name
pBudCE4.1-hrGFP,Plasmid pBudCE4.1-hrGFP,pBudCE4.1-hrGFP vector
pBudCE4.1-hrGFP Informaiton
Promoter: CMV promoter, EF1- alpha promoter
Replicator: SV40 ori, ori
Terminator: bGH poly (A) signal
Plasmid classification: lactation serial plasmid; lactation expression plasmid; lactation bbox plasmid.
Plasmid size: 5303bp
Plasmid label: C-His, C-V5
Prokaryotic resistance: bleomycin Zeo (50 mu g/ml)
Screening markers: bleomycin Zeo
Cloned strains of Escherichia coli, DH5 A and other Escherichia coli
Culture conditions: 37 centigrade, aerobic, LB
Expression host: mammalian cells such as 293T
Culture conditions: 37 C, 5%CO2
Induction mode: no induction, instantaneous expression
Primers for 5'sequencing: CMV-F (CGCAAATGGGCGGTAGGCGTG) EF-1a- F (TCAAGCCTCAGACAGTGGTTC)
3'sequencing primers: Bleo-R (CTGGATGATCCTCCAGCGC) BGH (TAGAAGGCACAGTCGAGG)
pBudCE4.1-hrGFP Description
pBudCE4.1-hrGFP plasmid was cloned into the hrGFP green fluorescent gene in pBudCE4.1 vector. When the pBudCE4.1 plasmid is used, it is best to use the strain without Tn5 gene, such as TOP10 and DH5 alpha T1, when E.coli clones are cloned. The best use of electrotransfer is to use TOP10, and the working concentration of bleomycin Zeocin recommends 25 u g/ml ~50 mu g/ml.
The pBudCE4.1 vector is designed to independently express two genes from a single vector in mammalian cells. The use of pBudCE4.1 to produce stable mammalian expression cell lines ensures that the copy number of each gene in the cell is equal. This can eliminate the change of expression caused by the difference of gene copy number. PBudCE4.1 can provide expression boxes with the following characteristics:
• CMV for high level transcription genes, which can be used for rapid detection of c-myc epitope tags and for simple purification of 6xHis sequences.
• EF-1 alpha promoters for highly expressed genes, which can be used for rapid detection of V5 epitope tags and for simple purification of 6xHis sequences.
Sh ble (ZeoR) gene for efficient selection of Zeocin in mammalian cells and E. coli.
pBudCE4.1 is a 4.6 kb vector designed for simultaneous expression of two genes in mammalian cell lines. The vector contains the human cytomegalovirus (CMV) immediate-early promoter and the human elongation factor 1α-subunit (EF-1α) promoter for high-level, constitutive, independent expression of two recombinant proteins (see page 13 for more information on the EF-1α promoter). Features of the vector allow detection and purification of expressed proteins (see pages 15–16) for more information). High-level stable and transient expression studies can be carried out in most mammalian cell types. In addition to the two promoters, the vector contains the following elements:
• C-terminal peptides encoding the myc (c-myc) epitope or the V5 epitope and a polyhistidine (6xHis) metal-binding tag for detection and purification of recombinant proteins
• Zeocin™ resistance gene for selection in E. coli and creation of stable, mammalian cell lines (Mulsant et al., 1988) (see pages 18–19 for more information)
• SV40 origin for episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g., COS7) pBudCE4.1/lacZ/CAT is included for use as a positive control for transfection, expression, and detection in the cell line of choice.
pBudCE4.1 is an improved version of pBudCE4. During construction of the original vector, an ATG was inadvertently created in the multiple cloning site (672–674 bp) 3′to the CMV promoter. Since it may interfere with proper translation of the cloned gene, this ATG was changed to ATT to create pBudCE4.1.
pBudCE4.1-hrGFP Sequence
LOCUS Exported 5303 bp ds-DNA circular SYN 21-July-2016
DEFINITION synthetic circular DNA
KEYWORDS pBudCE4.1-hrGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5303)
TITLE Direct Submission
JOURNAL Exported 2016-09-18
FEATURES Location/Qualifiers
source 1..5303
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 10..389
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 390..593
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 638..656
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 676..1395
/codon_start=1
/product="humanized Renilla green fluorescent protein"
/note="hrGFP"
/note="mammalian codon-optimized"
/translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
DGGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
AQLTSLGKPLGSLHEWV"
CDS 1427..1456
/codon_start=1
/product="Myc (human c-Myc oncogene) epitope tag"
/note="Myc"
/translation="EQKLISEEDL"
CDS 1472..1489
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
polyA_signal 1520..1641
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
CDS complement(1771..2145)
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/note="BleoR"
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
promoter complement(2164..2211)
/note="EM7 promoter"
/note="synthetic bacterial promoter "
promoter complement(2259..2575)
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2273..2408
/note="SV40 ori"
/note="SV40 origin of replication"
promoter 2597..3759
/note="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2828..3750
/note="EF-1-alpha intron A"
/note="intron upstream of the start codon of human
EF-1-alpha"
CDS 3835..3876
/codon_start=1
/product="epitope tag from simian virus 5"
/note="V5 tag"
/translation="GKPIPNPLLGLDST"
CDS 3886..3903
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
polyA_signal 3932..4156
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin complement(4288..4876)
