The pTYB1 is an E. coli vector created for fusion protein expression and purification. A target gene can be inserted into the the ORF by use of the MCS upstream of the Sce VMA intein. The NdeI site in the MCS has the initiator sequence ATG that enables translation of the ORF. The chitin-binding domain(CBD) downstream of the intein results in a target protein-intein with C-terminal chitin beads that facilitate protein purification. Release of the protein is induced by the thiol cleavage activity of the intein.
| 载体基本信息 | |||
| 载体名称 | pTYB1 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 7477 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 | 载体标签 | Intein |
pTYB1 载体载体图谱
pTYB1 载体载体序列
LOCUS Exported 7477 bp ds-DNA circular SYN 18-SEP-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pTYB1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7477)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7477
/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 5776..6594
/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 7050..7061
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
CDS 7171..7326
/codon_start=1
/gene="Bacillus circulans chiA"
/product="chitin binding domain from chitinase A1 (Watanabe
et al., 1994)"
/note="CBD"
/translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
PALWQLQ"
terminator 7405..7452
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
