pTYB2 is an E. coli cloning and expression vector (7474 bp) used in the IMPACT™ Kit (NEB #E6901) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag (1,2). This C-terminal fusion vector is designed for the in-frame insertion of a target gene into the polylinker upstream of an intein tag (the Sce VMA intein/chitin binding domain, 55 kDa) (1,2). This results in the fusion of the C-terminus of the target protein to the N-terminus of the intein tag. Thiol-induced self-cleavage of the intein releases the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein. For fusion of the N-terminus of the target protein to the intein tag, use pTYB21 (NEB #N6709) or pTYB22 (NEB #N6710). This vector can be used in conjuction with pTYB22 (NEB #N6710) to test which fusion constructin (N-terminal or C-terminal) maximizes the expression and yield of a target protein (3).
| 载体基本信息 | |||
| 载体名称 | pTYB2 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 7474 bp | 载体类型 | E.coli expression plasmid |
| 拷贝数 | High copy number | 启动子 | T7 |
| 克隆方法 | Enzyme Cut | ||
pTYB2 载体载体图谱
pTYB2 载体载体序列
LOCUS Exported 7474 bp ds-DNA circular SYN 18-SEP-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pTYB2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7474)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7474
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 35..139
/gene="bla"
/note="AmpR promoter"
CDS 140..1000
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin complement(1042..1555)
/direction=LEFT
/note="M13 ori"
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
rep_origin 1666..2254
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2623..2814)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(3371..4453)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(4454..4531)
/gene="lacI"
/note="lacI promoter"
terminator 4684..4770
/gene="Escherichia coli rrnB"
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 4867..4953
/gene="Escherichia coli rrnB"
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5050..5136
/gene="Escherichia coli rrnB"
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5233..5319
/gene="Escherichia coli rrnB"
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5416..5502
/gene="Escherichia coli rrnB"
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
promoter 5637..5655
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 5656..5680
/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)."
RBS 5695..5717
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5773..6591
/codon_start=1
/gene="Saccharomyces cerevisiae VMA1"
/product="5 region of the intein from the yeast Vma1
subunit of the vacuolar ATPase (Chong et al., 1998)"
/note="Sce VMA intein 5 region"
/translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
CDS 7047..7058
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
CDS 7168..7323
/codon_start=1
/gene="Bacillus circulans chiA"
/product="chitin binding domain from chitinase A1 (Watanabe
et al., 1994)"
/note="CBD"
/translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
PALWQLQ"
terminator 7402..7449
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
