A Tetracycline-Regulated Expression System without Viral Transactivators The T-REx System yields higher levels of induced expression than any other regulated mammalian expression system. It utilizes the complete CMV promoter and adds control elements from the bacterial tetracycline resistance operon to effectively repress and derepress transcription from one of the strongest mammalian promoter sequences known (1,2).Specific Activation The T-REx System uses a repressor mechanism that blocks transcription from the powerful CMV promoter in the absence of tetracycline. Because the T-REx System elements do not use viral transactivators, you can achieve high-level expression from the complete CMV promoter without secondary, non-specific activation of host genes.The T-REx Mechanism The T-REx transcriptional control elements are illustrated in Figure 1. Two tetracycline operator sequences (TetO2) have been inserted between the TATA box of the CMV promoter and the
| 载体基本信息 | |||
| 载体名称 | pcDNA4/TO/Myc-His /LacZ | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 8198 bp | 载体类型 | Tetracycline Regulatory System |
| 筛选标记 | Zeocin | 启动子 | CMV |
| 克隆方法 | Enzyme digestion and ligation | 载体标签 | His Tag (6x), c-Myc Epitope Tag |
pcDNA4/TO/Myc-His /LacZ 载体载体图谱
pcDNA4/TO/Myc-His /LacZ 载体载体序列
LOCUS Exported 8198 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 8198)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8198
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 235..614
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 820..838
/gene="tetO"
/bound_moiety="tetracycline repressor TetR"
/note="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 841..859
/gene="tetO"
/bound_moiety="tetracycline repressor TetR"
/note="tet operator"
/note="bacterial operator O2 for the tetR and tetA genes"
CDS 1051..4095
/codon_start=1
/gene="lacZ"
/product="beta-galactosidase"
/note="lacZ"
/translation="VVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRS
LNGEWRFAWFPVPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNVTYPITVNPP
FVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYGQDSRLPSEF
DLSAFLRAGENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHKPTTQISDFHVATR
FNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGGEIIDERGGYADRV
TLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFREVRIENGLLLLNGKP
LLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNFNAVRCSHYPNHPLWYTLCDRYGL
YVVDEANIETHGMVPMNRLTDDPRWLPAMSERVTRMVQRDRNHPSVIIWSLGNESGHGA
NHDALYRWIKSVDPSRPVQYEGGGADTTATDIICPMYARVDEDQPFPAVPKWSIKKWLS
LPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQYPRLQGGFVWDWVDQSLIKYDENGNP
WSAYGGDFGDTPNDRQFCMNGLVFADRTPHPALTEAKHQQQFFQFRLSGQTIEVTSEYL
FRHSDNELLHWMVALDGKPLASGEVPLDVAPQGKQLIELPELPQPESAGQLWLTVRVVQ
PNATAWSEAGHISAWQQWRLAENLSVTLPAASHAIPHLTTSEMDFCIELGNKRWQFNRQ
SGFLSQMWIGDKKQLLTPLRDQFTRAPLDNDIGVSEATRIDPNAWVERWKAAGHYQAEA
ALLQCTADTLADAVLITTAHAWQHQGKTLFISRKTYRIDGSGQMAITVDVEVASDTPHP
ARIGLNCQLAQVAERVNWLGLGPQENYPDRLTAACFDRWDLPLSDMYTPYVFPSENGLR
CGTRELNYGPHQWRGDFQFNISRYSQQQLMETSHRHLLHAEEGTWLNIDGFHMGIGGDD
SWSPSVSAEFQLSAGRYHYQLVWCQK"
CDS 4120..4149
/codon_start=1
/product="Myc (human c-Myc oncogene) epitope tag"
/note="Myc"
/translation="EQKLISEEDL"
CDS 4165..4182
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
polyA_signal 4211..4435
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 4481..4909
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4923..5253
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 5104..5239
/note="SV40 ori"
/note="SV40 origin of replication"
promoter 5301..5348
/note="EM7 promoter"
/note="synthetic bacterial promoter "
CDS 5367..5741
/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"
polyA_signal 5871..5992
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(6041..6057)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 6065..6081
/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)."
promoter complement(6089..6119)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 6134..6155
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(6443..7031)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7202..8062)
/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"
promoter complement(8063..8167)
/gene="bla"
/note="AmpR promoter"
