pCMS-EGFP is a mammalian expression vector that allows you to express your gene of interest at high levels by cloning it into a multiple cloning site (MCS) downstream of the immediate early promoter of cytomegalovirus (PCMV IE). The intervening sequence (IVS) between PCMV IE and the MCS is an intron that is efficiently spliced out following transcription. SV40 polyadenylation signals downstream of the MCS direct proper processing of the 3 end of the mRNA from your gene of interest. Bacteriophage T7 and T3 promoters are located upstream and downstream of the MCS, respectively.pCMS-EGFP uses the enhanced green flourescent protein (EGFP) as a transformation marker. EGFP is a red-shifted variant of wild-type GFP from Aquorea victoria (1–3). EGFP has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) EGFP encodes the GFPmut1 variant (4) which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences (5). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (6) to further increase the translation efficiency in eukaryotic cells. EGFP is expressed from the SV40 enhancer/promoter, and a polyadenylation signal from the bovine growth hormone (BGH) gene directs proper processing of the 3 end of the EGFP mRNA. The SV40 origin also allows for replication in mammalian cells expressing the SV40 T antigen.The vector backbone also contains the β-lactamase gene for ampicillin resistance and a ColE1 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.The name of the vector comes from the order of four critical elements: the CMV IE promoter; the MCS; the SV40 promoter; and EGFP.Genes cloned into the MCS must contain an ATG start codon. pCMS-EGFP and derivatives can be introduced into mammalian cells by any standard method. Transfected cells can be monitored and/or selected by flow cytometry based on the fluorescence of EGFP. Sense or antisense RNA can be transcribed from the T7 and T3 promoters, respectively.
| 载体基本信息 | |||
| 载体名称 | pCMS-EGFP | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 5541 bp | 载体类型 | Fluorescent Protein Reporter Vectors |
| 启动子 | CMV | 克隆方法 | Enzyme digestion and ligation |
| 载体标签 | EGFP | ||
pCMS-EGFP 载体载体图谱
pCMS-EGFP 载体载体序列
LOCUS Exported 5541 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5541)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5541
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 138..517
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 518..729
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
intron 890..1022
/note="chimeric intron"
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
promoter 1067..1085
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter complement(1141..1158)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
polyA_signal 1174..1295
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 1481..1936
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2055..2412
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2263..2398
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2449..3168
/codon_start=1
/product="enhanced GFP"
/note="EGFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 3200..3424
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
promoter 3744..3848
/gene="bla"
/note="AmpR promoter"
CDS 3849..4709
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 4880..5468
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
