The pSV-β-Galactosidase Control Vector(a) is designed as a positive control vector for monitoring transfection efficiencies of mammalian cells. The SV40 early promoter and enhancer drive transcription of the bacterial lacZ gene, which in turn, is translated into the β-galactosidase enzyme. β-galactosidase is an excellent reporter enzyme (1,2), that can be assayed quickly and directly in cell extracts using spectrophotometric, fluorescent or chemiluminescent assays (3,4). This reporter enzyme is also widely used for in situ histochemical analysis using the substrate X-Gal (5).The pSV-β-Galactosidase Control Vector can be co-transfected with your DNA of interest. For example, co-transfection with firefly luciferase gene vectors (pGL3 Vectors) provide cell extracts that can be assayed for both luciferase and β-galactosidase activities. In this manner, the pSV-β-Galactosidase Vector acts as an internal control for transient expression assays. A negative control extract, prepared from mock-transfected cells, should also be assayed for the presence of endogenous β-galactosidase activity in cultured cells (2). In addition, co-transfection with chloramphenicol acetyltransferse reporter gene vectors (pCAT3 Vectors) permits assaying for both CAT and β-galactosidase activities.The pSV-β-Galactosidase Vector is a modification of pRSV-βGAL (6) with SV40 and pUC18 sequences substituted for RSV and pBR322 sequences. The pSV-β- Galactosidase Vector will express β-galactosidase in E. coli due to the presence of the E. coli gpt promoter located upstream of the lacZ gene (1). Colonies of E. coli containing the pSV-β-Galactosidase Vector will appear blue when plated on media containing X-gal.
| 载体基本信息 | |||
| 载体名称 | pSV-β-Galactosidase | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 6820 bp | 载体类型 | Mammalian Cell Expression Vectors |
| 克隆方法 | Enzyme digestion and ligation | ||
pSV-β-Galactosidase 载体载体图谱
pSV-β-Galactosidase 载体载体序列
LOCUS Exported 6820 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 6820)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..6820
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 56..413
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 264..399
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 710..3757
/codon_start=1
/gene="lacZ"
/product="beta-galactosidase"
/note="lacZ"
/translation="VVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRS
LNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNVTYPITVNPP
FVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYGQDSRLPSEF
DLSAFLRAGENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHKPTTQISDFHVATR
FNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGGEIIDERGGYADRV
TLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFREVRIENGLLLLNGKP
LLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNFNAVRCSHYPNHPLWYTLCDRYGL
YVVDEANIETHGMVPMNRLTDDPRWLPAMSERVTRMVQRDRNHPSVIIWSLGNESGHGA
NHDALYRWIKSVDPSRPVQYEGGGADTTATDIICPMYARVDEDQPFPAVPKWSIKKWLS
LPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQYPRLQGGFVWDWVDQSLIKYDENGNP
WSAYGGDFGDTPNDRQFCMNGLVFADRTPHPALTEAKHQQQFFQFRLSGQTIEVTSEYL
FRHSDNELLHWMVALDGKPLASGEVPLDVAPQGKQLIELPELPQPESAGQLWLTVRVVQ
PNATAWSEAGHISAWQQWRLAENLSVTLPAASHAIPHLTTSEMDFCIELGNKRWQFNRQ
SGFLSQMWIGDKKQLLTPLRDQFTRAPLDNDIGVSEATRIDPNAWVERWKAAGHYQAEA
ALLQCTADTLADAVLITTAHAWQHQGKTLFISRKTYRIDGSGQMAITVDVEVASDTPHP
ARIGLNCQLAQVAERVNWLGLGPQENYPDRLTAACFDRWDLPLSDMYTPYVFPSENGLR
CGTRELNYGPHQWRGDFQFNISRYSQQQLMETSHRHLLHAEEGTWLNIDGFHMGIGGDD
SWSPSVSAEFQLSAGRYHYQLVWCQK"
polyA_signal 4021..4155
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(4189..4205)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 4679..4783
/gene="bla"
/note="AmpR promoter"
CDS 4784..5644
/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 5815..6403
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 6691..6712
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6727..6757
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 6765..6781
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/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 6789..6805
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
