pBudCE4.1- hrGFP/ pBudCE4.1- hrGFP质粒/ pBudCE4.1- hrGFP Plasmid

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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)