pET28a- EGFP/ pET28a- EGFP质粒/ pET28a- EGFP Plasmid

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pET28a-EGFP Informaiton

Promoter: T7/lac

Copier: ColE1 ori, F1 ori

Terminator: T7 Terminator

Plasmid Classification: Colon Series Plasmids; Colon Fluorescent Plasmids; Colon Green Plasmids

Plasmid size: 6050bp

Plasmid tags: N-6 *His, N-Thrombin, N-EGFP, C-6 *His

Prokaryotic resistance: kanamycin Kan (50 ug/ml)

Cloning strains: Escherichia coli such as DH5alpha

Culture conditions: 37 C, aerobic, LB

Expression host: Escherichia coli such as BL21 (DE3)

Culture conditions: 37 C, aerobic, LB

Induction: IPTG or lactose and its analogues

5'Sequencing Primer: T7 (TAATACGACTCACTATAGG)

3'Sequencing primers: T7-ter (TGCTAGTTATTGCTCAGCGG)

pET28a-EGFP Description

pET28a-EGFP is a prokaryotic expression vector. The C-terminal contains a 6*His tag, and the N-terminal contains a 6*His tag, thrombin digestion site and GFP tag. The plasmid contains several commonly used restriction sites, which facilitates the cloning of different genes. The expression was induced by T7 RNA polymerase provided by host cells. The target gene was cloned into plasmid vector and controlled by strong transcription and translation signals of bacteriophages.

PET system is the most powerful system to express recombinant proteins in E. coli, and it is also the most widely used system in prokaryotic expression nowadays. This series of plasmids can easily weaken the expression of protein by reducing the concentration of inducers. Under non-induced conditions, the target gene can be completely silenced without transcription

pET28a-EGFP Multiple cloning sites

pET28a-EGFP Sequence

LOCUS       Exported                6050 bp ds-DNA     circular SYN 12-JUL-2017
DEFINITION  synthetic circular DNA
KEYWORDS    pET28a-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6050)
  TITLE     Direct Submission
  JOURNAL   Exported 2015-4-17  
REFERENCE   2  (bases 1 to 6050)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, July 12, 2017  
FEATURES             Location/Qualifiers
     source          1..6050
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      12..467
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 ori
                     f1 bacteriophage origin of replication; arrow indicates
                     direction of (+) strand synthesis"
     CDS             complement(560..1375)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /note="aph(3')-Ia"
                     /note="KanR
                     confers resistance to kanamycin in bacteria or G418
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      1497..2085
                     /direction=RIGHT
                     /note="ori"
                     /note="ori
                     high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     misc_feature    2271..2413
                     /note="bom"
                     /note="bom
                     basis of mobility region from pBR322"
     CDS             complement(2515..2706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3515..4597)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="lacI"
                     /note="lacI
                     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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(4598..4675)
                     /gene="lacI"
                     /note="lacI lacI promoter"
                     /note="lacI promoter"
     promoter        4984..5002
                     /note="T7 promoter"
                     /note="T7 promoter
                     promoter for bacteriophage T7 RNA polymerase"
     CDS             5083..5100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             5131..5847
                     /codon_start=1
                     /product="enhanced GFP"
                     /note="enhanced GFP"
                     /note="EGFP
                     mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
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