| 出品公司: | NEB |
|---|---|
| 载体名称: | pTWIN2 |
| 质粒类型: | 大肠杆菌表达载体 |
| 表达水平: | 高 |
| 启动子: | T7 |
| 克隆方法: | 多克隆位点，限制性内切酶 |
| 载体大小: | 7192 bp |
| 5' 测序引物及序列: | T7:TAATACGACTCACTATAGGG |
| 3' 测序引物及序列: | -- |
| 载体标签: | -- |
| 载体抗性: | Ampicillin |
| 筛选标记: | -- |
| 备注: | -- |
| 产品目录号: | E6901 |
| 稳定性: | -- |
| 组成型: | -- |
| 病毒/非病毒: | 非病毒 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
pTWIN2 is an E. coli expression vector which can be used with the IMPACT? Kit (NEB #E6901) (1). A polylinker in the vector is designed for the in-frame fusion of a target gene between the modified Ssp DnaB (2) and Mth RIR1 inteins (3). The presence of the chitin binding domain from Bacillus circulans (4,5) 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 (1). The double-stranded vector is 7192 base pairs in length.
载体序列
LOCUS pTWIN2 7192 bp DNA circular 09-NOV-2011
DEFINITION Cloning vector pTWIN2, complete sequence.
ACCESSION
VERSION
KEYWORDS .
SOURCE Cloning vector pTWIN2
ORGANISM Cloning vector pTWIN2
other sequences; artificial sequences; vectors.
REFERENCE 1 (bases 1 to 7192)
AUTHORS Evans,T.C. Jr., Benner,J. and 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,T.C. Jr., Chute,I.C., Wu,H., Chong,S., Benner,J.,
Liu,X.-Q. and 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,T.C. Jr., Benner,J. and 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,F.B.
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. and Xu,M.Q.
TITLE Direct Submission
JOURNAL Submitted (23-OCT-2007) Research Department, New England Biolabs,
240 County Road, Ipswich, MA 01938, USA
FEATURES Location/Qualifiers
source 1..7192
/organism="Cloning vector pTWIN2"
/mol_type="other DNA"
gene 140..1000
/gene="bla"
CDS 140..1000
/gene="bla"
/note="ampR (confers resistance to ampicillin)"
/codon_start=1
/product="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERSPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW"
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)"
gene complement(2623..2814)
/gene="rop"
CDS complement(2623..2814)
/gene="rop"
/exception="alternative start codon"
/codon_start=1
/transl_table=11
/product="ROP protein"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH
ADELYRSCLARFGDDGENL"
gene complement(3371..4453)
/gene="lacI"
CDS complement(3371..4453)
/gene="lacI"
/exception="alternative start codon"
/codon_start=1
/transl_table=11
/product="lac repressor (LacI)"
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE
LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS
GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG
DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT
EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT
QTASPRALADSLMQLARQVSRLESGQ"
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
/note="lac operator"
/bound_moiety="LacI"
RBS 5710..5716
/note="RBS for expression CDS"
gene 5725..7044
/gene="expression CDS"
CDS 5725..7044
/gene="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"
/codon_start=1
/product="CBD/intein/intein/CBD fusion"
/translation="MKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSL
AGWEPSNVPALWQLQNNGNNGLELRESGAISGDSLISLASTGKRVSIKDLLDEKDFEI
WAINEQTMKLESAKVSRVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDELS
LKEHIALPRKLESSSLQLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFV
ANDIIVHNGRAMGGREFLEGSSCVSGDTIVMTSGGPRTVAELEGKPFTALIRGSGYPC
PSGFFRTCERDVYDLRTREGHCLRLTHDHRVLVMDGGLEWRAAGELERGDRLVMDDAA
GEFPALATFRGLRGAGRQDVYDATVYGASAFTANGFIVHACGEQPGTGLNSGLTTNPG
VSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ"
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"
BASE COUNT 1785 a 1803 c 1934 g 1670 t
ORIGIN
1 aactacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
61 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
121 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
181 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
241 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
301 ccttgagagt tttcgccccg aagaacgttc tccaatgatg agcactttta aagttctgct
361 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
421 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
481 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
541 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
601 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
661 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
721 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
781 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
841 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
901 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
961 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
1021 atatatactt tagattgatt taccccggtt gataatcaga aaagccccaa aaacaggaag
1081 attgtataag caaatattta aattgtaaac gttaatattt tgttaaaatt cgcgttaaat
1141 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
1201 tcaaaagaat agcccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
1261 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
1321 ctacgtgaac catcacccaa atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
1381 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
1441 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
1501 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtaaaag
1561 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
1621 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
1681 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
1741 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
1801 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
1861 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
1921 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
1981 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
2041 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
2101 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
2161 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
2221 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
2281 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
2341 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
2401 cgagcgcagc gagtcagtga gcgaggaagc tatggtgcac tctcagtaca atctgctctg
2461 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
2521 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
2581 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
2641 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gcagcgattc
2701 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
2761 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
2821 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
2881 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
2941 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcc
3001 gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt
3061 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
3121 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
3181 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
3241 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
3301 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
3361 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
3421 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
3481 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
3541 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
3601 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
3661 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
3721 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
3781 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
3841 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
3901 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
3961 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
4021 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
4081 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
4141 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
4201 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
4261 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
4321 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
4381 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
4441 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
4501 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
4561 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
4621 aatggtgcat gccggcatgc cgccctttcg tcttcaagaa ttaattccca attccccagg
4681 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
4741 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag
4801 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggaa ttaattcccc
4861 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
4921 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
4981 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gaattaattc
5041 cccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
5101 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt
5161 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggaattaa
5221 ttccccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
5281 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac
5341 gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggaat
5401 taattcccca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
5461 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
5521 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
5581 aattggggat cggaattaat tcccggttta aaccggggat ctcgatcccg cgaaattaat
5641 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
5701 taactttaag aaggagatat acatatgaaa atcgaagaag gtaaactgac aaatcctggt
5761 gtatccgctt ggcaggtcaa cacagcttat actgcgggac aattggtcac atataacggc
5821 aagacgtata aatgtttgca gccccacacc tccttggcag gatgggaacc atccaacgtt
5881 cctgccttgt ggcagcttca aaacaacggt aacaacggtc tcgaactgcg cgagtccgga
5941 gctatctctg gcgatagtct gatcagcctg gctagcacag gaaaaagagt ttctattaaa
6001 gatttgttag atgaaaaaga ttttgaaata tgggcaatta atgaacagac gatgaagcta
6061 gaatcagcta aagttagtcg tgtattttgt actggcaaaa agctagttta tattctaaaa
6121 actcgactag gtagaactat caaggcaaca gcaaatcata gatttttaac tattgatggt
6181 tggaaaagat tagatgagct atctttaaaa gagcatattg ctctaccccg taaactagaa
6241 agctcctctt tacaattgtc accagaaata gaaaagttgt ctcagagtga tatttactgg
6301 gactccatcg tttctattac ggagactgga gtcgaagagg tttttgattt gactgtgcca
6361 ggaccacata actttgtcgc gaatgacatc attgtacaca acggaagagc catgggcggc
6421 cgcgaattcc tcgagggctc ttcctgcgta tccggtgaca ccattgtaat gactagtggc
6481 ggtccgcgca ctgtggctga actggagggc aaaccgttca ccgcactgat tcgcggctct
6541 ggctacccat gcccctcagg tttcttccgc acctgtgaac gtgacgtata tgatctgcgt
6601 acacgtgagg gtcattgctt acgtttgacc catgatcacc gtgttctggt gatggatggt
6661 ggcctggaat ggcgtgccgc gggtgaactg gaacgcggcg accgcctggt gatggatgat
6721 gcagctggcg agtttccggc actggcaacc ttccgtggcc tgcgtggcgc tggccgccag
6781 gatgtttatg acgctactgt ttacggtgct agcgcattca ctgctaatgg cttcattgta
6841 cacgcatgcg gcgagcagcc cgggaccggt ctgaactcag gcctcacgac aaatcctggt
6901 gtatccgctt ggcaggtcaa cacagcttat actgcgggac aattggtcac atataacggc
6961 aagacgtata aatgtttgca gccccacacc tccttggcag gatgggaacc atccaacgtt
7021 cctgccttgt ggcagcttca atgactgcag gaaggggatc cggctgctaa caaagcccga
7081 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
7141 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg at
//
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