pTYB12 is an E. coli cloning and expression vector (7415 bp) used in the IMPACT™ Protein Purification System which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag (1,2). It is a N-terminal fusion vector designed for in-frame insertion of a target gene into the polylinker, downstream of the intein tag (the Sce VMA intein/chitin domain, 55 kDa)(3,4). This allows the N-terminus of the target protein to be fused to the intein tag. The self-cleavage activity of the intein allows the release of the target protein from the chitin-bound intein tag, resulting in a single column purification of the target protein. This vector can be used in conjuction with pTYB2 (NEB #N6702S) to test which fusion construction (N-terminal or C-terminal) maximizes the expression and yield of a target protein.
| 载体基本信息 | |||
| 载体名称 | pTYB12 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 7415 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 | ||
pTYB12 载体载体图谱
pTYB12 载体载体序列
LOCUS Exported 7415 bp ds-DNA circular SYN 18-SEP-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7415)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7415
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 1..28
/note="rrnB T2 terminator"
/note="transcription terminator T2 from the E. coli rrnB
gene"
promoter 48..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"
promoter 4840..4858
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 4859..4883
/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 4898..4920
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 4973..5791
/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 5798..5950
/codon_start=1
/gene="Bacillus circulans chiA"
/product="chitin binding domain from chitinase A1 (Watanabe
et al., 1994)"
/note="CBD"
/translation="TNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVP
ALWQLQ"
CDS 5954..6499
/codon_start=1
/gene="Saccharomyces cerevisiae VMA1"
/product="modified 3 region of the intein from the yeast
Vma1 subunit of the vacuolar ATPase (Chong et al., 1998)"
/note="Sce VMA intein 3 region"
/note="C-terminal cleavage of this intein can be induced by
thiols."
/translation="GHGGIRNNLNTENPLWDAIVGLGFLKDGVKNIPSFLSTDNIGTRE
TFLAGLIDSDGYVTDEHGIKATIKTIHTSVRDGLVSLARSLGLVVSVNAEPAKVDMNVT
KHKISYAIYMSGGDVLLNVLSKCAGSKKFRPAPAAAFARECRGFYFELQELKEDDYYGI
TLSDDSDHQFLLGSQVVVQ"
terminator 6638..6685
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
primer_bind complement(6873..6889)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
terminator 7238..7324
/gene="Escherichia coli rrnB"
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
