The pcDNA6⁄TR vector is part of the T-REx System, which 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.The regulatory vector, pcDNA6⁄TR, is provided for high-level expression of the tetracycline repressor (TR) protein. This vector expresses the Blasticidin resistance gene for rapid selection of mammalian cell lines that stably express the TR protein.
| 载体基本信息 | |||
| 载体名称 | pcDNA6/TR | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 6662 bp | 载体类型 | Tetracycline Regulatory System |
| 筛选标记 | Blasticidin | 启动子 | CMV |
| 克隆方法 | Enzyme digestion and ligation | 5引物 | pCAG-F; Bglob-intron-F |
| 3引物 | EBV-rev; BGH-rev | ||
pcDNA6/TR 载体载体图谱
pcDNA6/TR 载体载体序列
LOCUS Exported 6662 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 6662)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..6662
/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"
promoter 863..881
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
intron 1028..1600
/note="beta-globin intron"
/note="intron from rabbit beta-globin gene"
promoter 1655..1673
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1684..2301
/codon_start=1
/gene="tetR from transposon Tn10"
/product="tetracycline repressor TetR"
/note="TetR"
/note="TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved by
adding tetracycline or doxycycline."
/translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
polyA_signal 2346..2467
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
CDS 2530..2571
/codon_start=1
/product="epitope tag from simian virus 5"
/note="V5 tag"
/translation="GKPIPNPLLGLDST"
CDS 2581..2598
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
polyA_signal 2627..2851
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 2897..3325
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3339..3668
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 3519..3654
/note="SV40 ori"
/note="SV40 origin of replication"
promoter 3716..3763
/note="EM7 promoter"
/note="synthetic bacterial promoter "
CDS 3782..4180
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/note="BSD"
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
polyA_signal 4338..4459
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(4508..4524)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 4532..4548
/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(4556..4586)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 4601..4622
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(4910..5495)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5666..6526)
/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(6527..6631)
/gene="bla"
/note="AmpR promoter"
