The pRDV is a ribosome-display vector. pRDV contains all flanking DNA regions necessary for ribosome display: the T7-promoter, the ribosomal binding site and an in-frame tolA gene spacer. Hence, by simple ligation of the DNA encoding the combinatorial library into pRDV and by a PCR using this ligation mix as template, all features necessary for ribosome display are added to the library. The use of pRDV has the advantage that it always provides error-free library flanking regions and that it saves a number of working steps compared to the standard PCR approach for library generation.
| 载体基本信息 | |||
| 载体名称 | pRDV | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 4279 bp | 载体类型 | Ribosome display vector |
| 载体来源 | Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, Grutter MG, Pluckthun A. | ||
pRDV 载体载体图谱
pRDV 载体载体序列
LOCUS Exported 4279 bp ds-DNA circular SYN 18-DEC-2018
DEFINITION Ribosome display vector pRDV, complete sequence.
ACCESSION AY327136
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4279)
AUTHORS Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, Grutter
MG, Pluckthun A.
TITLE High-affinity binders selected from designed ankyrin repeat protein
libraries
JOURNAL Nat. Biotechnol. 22 (5), 575-582 (2004)
PUBMED 15097997
REFERENCE 2 (bases 1 to 4279)
AUTHORS Amstutz P, Zahnd C, Binz KH, Stumpp MT, Forrer P, Pluckthun A.
TITLE Direct Submission
JOURNAL Submitted (20-JUN-2003) Biochemistry, University of Zurich,
Winterthurerstrasse 190, Zurich 8057, Switzerland
REFERENCE 3 (bases 1 to 4279)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4279
/organism="Ribosome display vector pRDV"
/mol_type="genomic DNA"
/db_xref="taxon:248461"
promoter 19..37
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 19..35
/regulatory_class="promoter"
/note="T7"
stem_loop 36..56
protein_bind 60..76
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
RBS 87..109
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
regulatory 103..108
/regulatory_class="ribosome_binding_site"
CDS 117..1862
/codon_start=1
/product="RDfusion"
/label=RDfusion
/note="Flag-tag_beta-lactamase_tolA fusion protein"
/protein_id="AAQ93652.1"
/translation="MADYKDDDDKGSHPETLVKVKDAEDQLGARVGYIELDLNSGKILE
SFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGM
TVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPND
ERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKS
GAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWGKLYMA
SGAEFGSGGQKQAEEAAAKAAADAKAKAEADAKAAEEAAKKAAADAKKKAEAEAAKAAA
EAQKKAEAAAAALKKKAEAAEAAAAEARKKAATEAAEKAKAEAEKKAAAEKAAADKKAA
AEKAAADKKAAEKAAAEKAAADKKAAAEKAAADKKAAAAKAAAEKAAAAKAAAEADDIF
GELSSGKNAPKTGGGAKGNNASPAGSGNTKNNGASGADINNYAGQIKSAIESKFYDASS
YAGKTCTLRIKLAPDGMLLDIKPEGGDPALCQAALAAAKLAKIPKPPSQAVYEVFKNAP
LDFKP"
misc_feature 117..146
/label=Flag-tag
/note="Flag-tag"
CDS 123..146
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
misc_feature 147..974
/label=beta-lactamase insert flanked by restriction sites
/note="beta-lactamase insert flanked by restriction sites"
CDS 156..941
/codon_start=1
/gene="bla(M)"
/product="beta-lactamase lacking the signal sequence"
/label=bla(M)
/note="allows cytosolic expression of beta-lactamase"
/translation="PETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMST
FKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS
DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATT
LRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAA
LGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
misc_feature 975..1862
/label=tolA spacer
/note="tolA spacer"
terminator 1930..1977
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2013..2468
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2495..2599
/gene="bla"
/label=AmpR promoter
CDS 2600..3460
/codon_start=1
/product="beta-lactamase"
/label=beta-lactamase
/note="ampicillin resistance"
/protein_id="AAQ93651.1"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 3616..4272
/label=ColE1
/note="ColE1"
rep_origin 3631..4219
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
