Basic Vector Information
pTWIN2 is an E. coli expression vector which can be used with the IMPACTâ„¢ 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.
Basic Vector Information | |||
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Vector Name | pTWIN2 | Antibiotic Resistance | Ampicillin |
Length | 7192 bp | Type | Bacterial expression vector |
Source | NEB | Copy Number | High copy number |
Promoter | T7 | Fusion Tag | intein |
Expression Method | IPTG induced |
pTWIN2 vector Vector Map
pTWIN2 vector Sequence
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 JOURNAL Exported Wednesday, December 9, 2020 from SnapGene 3.2.1 http://www.snapgene.com 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"
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