pTWIN1 is an E. coli plasmid cloning vector designed for recombinant protein expression, labeling, and cyclization using the IMPACT-TWIN Kit (NEB #E6901) (1). It contains the pMB1 origin of replication from pBR322 and is maintained at a similar copy number to pBR322; in addition, pTWIN1 also contains an M13 origin of replication. The multiple cloning site (MCS) is positioned to allow translational fusion of an intein tag to the N-terminus, C-terminus, or both, of the cloned target protein. The mini-inteins encoded by pTWIN1, the Ssp DnaB intein and the Mxe GyrA intein, cleave the peptide bond at their C- and N-termini, respectively (1-4). The chitin binding domain (CBD) from B. circulans, fused to each intein, facilitates purification of the intein-target protein precursor. Transcription of the intein tags is controlled by the inducible T7 promoter, requiring E. coli strains containing integrated copies of the T7 RNA polymerase gene [e.g., NEB #C2566, #C2833 or BL21(DE3)] for expression. Basal expression from the T7 promoter is minimized by the binding of the Lac repressor, encoded by the lacI gene, to the lac operator immediately downstream of the T7 promoter (5). Translation of the intein tags utilizes the translation initiation signal (Shine Dalgarno sequence) from the strongly expressed T7 gene 10 protein (φ10).
| 载体基本信息 | |||
| 载体名称 | pTWIN1 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 7375 bp | 载体类型 | Bacterial expression vector |
| 载体来源 | NEB | 拷贝数 | High copy number |
| 启动子 | T7 | 克隆方法 | Enzyme Cut |
| 载体标签 | intein | 表达方法 | IPTG induced |
pTWIN1 载体载体图谱
pTWIN1 载体载体序列
LOCUS Exported 7375 bp ds-DNA circular SYN 09-DEC-2020
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pTWIN1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7375)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7375
/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 5725..5901
/codon_start=1
/gene="Bacillus circulans chiA"
/product="chitin binding domain from chitinase A1 (Watanabe
et al., 1994)"
/note="CBD"
/translation="MKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLA
GWEPSNVPALWQLQ"
CDS 5941..6402
/codon_start=1
/note="Ssp DnaB intein"
/translation="AISGDSLISLASTGKRVSIKDLLDEKDFEIWAINEQTMKLESAKV
SRVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDELSLKEHIALPRKLESSSL
QLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFVANDIIVHN"
CDS 6445..7038
/codon_start=1
/gene="Mycobacterium xenopi gyrA"
/product="modified GyrA intein (Southworth et al., 1999)"
/note="Mxe GyrA intein"
/note="N-terminal cleavage of this intein can be induced by
thiols."
/translation="CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPV
LADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAV
IQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYY
AKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHA"
CDS 7069..7224
/codon_start=1
/gene="Bacillus circulans chiA"
/product="chitin binding domain from chitinase A1 (Watanabe
et al., 1994)"
/note="CBD"
/translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
PALWQLQ"
terminator 7303..7350
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
