pTWIN2 is an E. coli expression vector which can be used with the IMPACT™ Kit. A polylinker in the vector is designed for the in-frame fusion of a target gene between the modified Ssp DnaB and Mth RIR1 inteins. The presence of the chitin binding domain from Bacillus circulans facilitates purification. pTWIN vectors are designed for protein purification or for the isolation of proteins with an N-terminal cysteine and/or a C-terminal thioester. The double-stranded vector is 7192 base pairs in length.
| 载体基本信息 | |||
| 载体名称 | pTWIN2 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 7192 bp | 载体类型 | Bacterial expression vector |
| 载体来源 | NEB | 拷贝数 | High copy number |
| 启动子 | T7 | 载体标签 | intein |
| 表达方法 | IPTG induced | ||
pTWIN2 载体载体图谱
pTWIN2 载体载体序列
LOCUS Exported 7192 bp ds-DNA circular SYN 09-DEC-2020
DEFINITION Cloning vector pTWIN2, complete sequence.
ACCESSION .
VERSION .
KEYWORDS pTWIN2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7192)
AUTHORS Evans TC Jr., Benner J, Xu M-Q.
TITLE The in vitro ligation of bacterially expressed protein using an
intein from Methanobacterium thermoautotrophicum
JOURNAL J Biol Chem 274, 3923-3926 (1999)
PUBMED 9933578
REFERENCE 2 (bases 1 to 7192)
AUTHORS Mathys S, Evans TC Jr., Chute IC, Wu H, Chong S, Benner J, Liu X-Q.,
Xu M-Q.
TITLE Characterization of a self-splicing mini-intein and its conversion
into autocatalytic N- and C-terminal cleavage elements: facile
production of protein building blocks for protein ligation
JOURNAL Gene 231, 1-13 (1999)
PUBMED 10231563
REFERENCE 3 (bases 1 to 7192)
AUTHORS Evans TC Jr., Benner J, Xu M-Q.
TITLE The cyclization and polymerization of bacterially-expressed proteins
using modified self-splicing inteins
JOURNAL J Biol Chem 274, 18359-18363 (1999)
PUBMED 10373440
REFERENCE 4 (bases 1 to 7192)
AUTHORS Perler FB.
TITLE InBase, the intein database
JOURNAL Nucleic Acids Res 30 (1), 383-384 (2002)
PUBMED 11752343
REFERENCE 5 (bases 1 to 7192)
AUTHORS Ghosh I, Xu MQ.
TITLE Direct Submission
JOURNAL Submitted (23-OCT-2007) Research Department, New England Biolabs,
240 County Road, Ipswich, MA 01938, USA
REFERENCE 6 (bases 1 to 7192)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7192
/organism="Cloning vector pTWIN2"
/mol_type="other DNA"
CDS 140..1000
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="bla"
/note="ampR (confers resistance to ampicillin)"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
gene 140..1000
/gene="bla"
/note="bla"
sig_peptide 140..208
/gene="bla"
/note="required for secretion to the periplasm; cleaved off
to form the mature beta-lactamase protein"
rep_origin 1042..1555
/note="M13 origin of replication (+ -)"
rep_origin 1666..2254
/note="pMB1 origin of replication (clockwise) (RNAII -35 to
RNA/DNA switch point)"
CDS complement(2623..2814)
/codon_start=1
/transl_table=11
/exception="alternative start codon"
/gene="rop"
/product="ROP protein"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
gene complement(2623..2814)
/gene="rop"
/note="rop"
CDS complement(3371..4453)
/codon_start=1
/transl_table=11
/exception="alternative start codon"
/gene="lacI"
/product="lac repressor (LacI)"
/note="lacI"
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
gene complement(3371..4453)
/gene="lacI"
/note="lacI"
primer_bind 5637..5656
/note="T7 universal primer sequence (NEB #S1248S)"
promoter 5637..5654
/note="T7 promoter (transcript start 5654 clockwise)"
protein_bind 5656..5680
/bound_moiety="LacI"
/note="LacI lac operator"
/note="lac operator"
RBS 5710..5716
/note="RBS for expression CDS"
CDS 5725..7044
/codon_start=1
/gene="expression CDS"
/product="CBD/intein/intein/CBD fusion"
/note="expression CDS"
/note="chitin binding domain (CBD) sequence 5725..5901; Ssp
DNAB intein sequence 5941..6402; multiple cloning site
(SapI-SapI) 6403..6444; Mth RIR1 intein sequence
6445..6846; CBD sequence 6886..7044"
/translation="MKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLA
GWEPSNVPALWQLQNNGNNGLELRESGAISGDSLISLASTGKRVSIKDLLDEKDFEIWA
INEQTMKLESAKVSRVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDELSLKE
HIALPRKLESSSLQLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFVANDI
IVHNGRAMGGREFLEGSSCVSGDTIVMTSGGPRTVAELEGKPFTALIRGSGYPCPSGFF
RTCERDVYDLRTREGHCLRLTHDHRVLVMDGGLEWRAAGELERGDRLVMDDAAGEFPAL
ATFRGLRGAGRQDVYDATVYGASAFTANGFIVHACGEQPGTGLNSGLTTNPGVSAWQVN
TAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ"
gene 5725..7044
/gene="expression CDS"
/note="expression CDS"
primer_bind 6296..6315
/note="Ssp DnaB intein forward primer sequence (NEB
#S1269S)"
misc_feature 6403..6444
/gene="expression CDS"
/note="multiple cloning site (SapI-SapI)"
primer_bind complement(6505..6522)
/note="Mth RIR1 intein reverse primer sequence (NEB
#S1270S)"
terminator 7064..7186
/note="T7 Tphi transcription terminator"
