pCold TF

pCold TF

pColdTF载体基本信息

出品公司:	Ybscience
载体名称:	pCold TF, pColdTF, pCold-TF
质粒类型:	大肠杆菌蛋白表达载体
表达水平:	高
启动子:	CSPA
克隆方法:	多克隆位点，限制性内切酶
载体大小:	5769 bp
5' 测序引物及序列:	pCold-TF-F1: 5'-CCACTTTCAACGAGCTGATG-3'; pCold-TF-F2: 5'-GCGAAAGTGACTGAAAAAG-3'
3' 测序引物及序列:	pCold-R: 5'-GGCAGGGATCTTAGATTCTG-3'
载体标签:	N-His, N-Trigger factor, N-HRV 3C, N-Thrombin, N-Factor Xa
载体抗性:	Ampicillin 氨苄
备注:	温度诱导，冷休克蛋白表达载体
稳定性:	瞬时表达 Transient
组成型:	诱导表达
病毒/非病毒:	非病毒

pColdTF载体质粒图谱和多克隆位点信息

pColdTF载体简介

Takara的pCold TF载体是一种融合表达可溶性的标签"触发因子（Trigger Factor,TF）伴侣"的冷休克载体。"触发因子"（48KD）是原核核糖体相关的分子伴侣蛋白（48 kDa），有利于新表达的多肽的共翻译折叠。正因为它来源于大肠杆菌，保证了触发因子能够在大肠杆菌表达系统中高表达。 pCold TF载体由CSPA启动子及其下游的序列组成，其下游序列主要包括包括5'非翻译区（5'非编码区），翻译增强元件（TEE），His标签序列，多克隆位点（MCS）。Lac操纵子插入在CSPA启动子下游，以确保严格的表达调控。此外，HRV 3C蛋白酶，凝血酶，和Factor Xa识别位点位于“Trigger Factor”和多克隆位点（MCS）之间，以便表达的融合蛋白上将标签去除。

大多数大肠杆菌菌株都可以可以作为本载体的表达宿主。pCold TF载体提供冷休克诱导的方法高水平表达目的蛋白，再加上触发因子（伴侣）的表达来提高正确的蛋白质折叠，从而实现高效可溶性表达其他系统中难以对付的蛋白。

pColdTF载体序列

LOCUS       pCold\TF                5769 bp    DNA     circular SYN 27-DEC-2006
DEFINITION  Expression vector pColdTF DNA, complete sequence.
ACCESSION   AB213654
VERSION     AB213654.1  GI:66472063
KEYWORDS    .
SOURCE      Expression vector pColdTF
  ORGANISM  Expression vector pColdTF
            other sequences; artificial sequences; vectors.
COMMENT     This file is created by Vector NTI
            http://www.biofeng.com/
COMMENT     VNTAUTHORNAME|biofeng.com|
FEATURES             Location/Qualifiers
     source          1..5769
                     /organism="Expression vector pColdTF"
                     /mol_type="other DNA"
                     /db_xref="taxon:329577"
                     /lab_host="Escherichia coli"
                     /note="expresses an open reading frame as a trigger factor
                     fusion, containing the translation enhancing element,
                     His6, protease cleavage site for HRV 3C protease, thrombin
                     and factor Xa"
     gene            join(15..255,256..270)
                     /gene="cspA"
     promoter        15..81
                     /gene="cspA"
     misc_feature    82..121
                     /gene="cspA"
                     /note="synthetic lac operator sequence"
     misc_feature    122..255
                     /gene="cspA"
                     /note="modified cspA 5'UTR"
     gene            256..1725
                     /gene="tig"
     CDS             256..1725
                     /gene="tig"
                     /codon_start=1
                     /transl_table=11
                     /product="trigger factor tag"
                     /protein_id="BAD98926.1"
                     /db_xref="GI:66472064"
                     /translation="MNHKVHHHHHHMQVSVETTQGLGRRVTITIAADSIETAVKSELV
                     NVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPT
                     YVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQA
                     TWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKA
                     GEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEG
                     LRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFG
                     GNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDP
                     KEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAGLEVL
                     FQGPSAGLVPRGSGGIEGRHMELGTLEGSEFKLVDLQSR"
     misc_feature    256..270
                     /gene="cspA"
                     /note="TEE (translation enhancing element)"
     misc_feature    271..288
                     /gene="tig"
                     /note="His6 tag"
     misc_feature    1594..1617
                     /gene="tig"
                     /note="HRV 3C Protease site"
     misc_feature    1627..1644
                     /gene="tig"
                     /note="Thrombin site"
     misc_feature    1651..1662
                     /gene="tig"
                     /note="Factor Xa site"
     misc_feature    1663..1722
                     /gene="tig"
                     /note="MCS = Multiple cloning sites contain the follow
                     restriction sites: NdeI, SacI, KpnI, XhoI, BamHI, EcoRI,
                     HindIII, SalI, PstI, XbaI"
     gene            1730..1874
                     /gene="cspA"
     misc_feature    1730..1874
                     /gene="cspA"
                     /note="cspA 3'UTR"
     misc_feature    complement(2073..2546)
                     /note="M13 intergenic region"
     gene            2733..3593
                     /gene="Amp"
     CDS             2733..3593
                     /gene="Amp"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="BAD98927.1"
                     /db_xref="GI:66472065"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
     rep_origin      3753..4367
                     /note="ColE1 origin"
     gene            complement(4524..5606)
                     /gene="lacI"
     CDS             complement(4524..5606)
                     /gene="lacI"
                     /codon_start=1
                     /transl_table=11
                     /product="lactose operon repressor"
                     /protein_id="BAD98928.1"
                     /db_xref="GI:66472066"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE
                     LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS
                     GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG
                     DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT
                     EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT
                     QTASPRALADSLMQLARQVSRLESGQ"
ORIGIN      
        1 aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa
       61 tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat
      121 ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt
      181 cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt
      241 aagaggtaat acaccatgaa tcacaaagtg catcatcatc atcatcacat gcaagtttca
      301 gttgaaacca ctcaaggcct tggccgccgt gtaacgatta ctatcgctgc tgacagcatc
      361 gagaccgctg ttaaaagcga gctggtcaac gttgcgaaaa aagtacgtat tgacggcttc
      421 cgcaagggca aagtgccaat gaatatcgtt gctcagcgtt atggcgcgtc tgtacgccag
      481 gacgttctgg gtgacctgat gagccgtaac ttcattgacg ccatcattaa agaaaaaatc
      541 aatccggctg gcgcaccgac ttatgttccg ggcgaataca agctgggtga agacttcact
      601 tactctgtag agtttgaagt ttatccggaa gttgaactgc aaggtctgga agcgatcgaa
      661 gttgaaaaac cgatcgttga agtgaccgac gctgacgttg acggcatgct ggatactctg
      721 cgtaaacagc aggcgacctg gaaagaaaaa gacggcgctg ttgaagcaga agaccgcgtg
      781 accatcgact tcaccggttc tgtagacggc gaagagttcg aaggcggtaa agcgtctgat
      841 ttcgtactgg cgatgggcca gggtcgtatg atcccgggct ttgaagacgg tatcaaaggc
      901 cacaaagctg gcgaagagtt caccatcgac gtgaccttcc cggaagaata ccacgcagaa
      961 aacctgaaag gtaaagcagc gaaattcgct atcaacctga agaaagttga agagcgtgaa
     1021 ctgccggaac tgaccgcaga gttcatcaaa cgtttcggcg ttgaagatgg ttccgtagaa
     1081 ggtctgcgcg ctgaagtgcg taaaaacatg gagcgcgagc tgaagagcgc catccgtaac
     1141 cgcgttaagt ctcaggcgat cgaaggtctg gtaaaagcta acgacatcga cgtaccggct
     1201 gcgctgatcg acagcgaaat cgacgttctg cgtcgccagg ctgcacagcg tttcggtggc
     1261 aacgaaaaac aagctctgga actgccgcgc gaactgttcg aagaacaggc taaacgccgc
     1321 gtagttgttg gcctgctgct gggcgaagtt atccgcacca acgagctgaa agctgacgaa
     1381 gagcgcgtga aaggcctgat cgaagagatg gcttctgcgt acgaagatcc gaaagaagtt
     1441 atcgagttct acagcaaaaa caaagaactg atggacaaca tgcgcaatgt tgctctggaa
     1501 gaacaggctg ttgaagctgt actggcgaaa gcgaaagtga ctgaaaaaga aaccactttc
     1561 aacgagctga tgaaccagca ggcgtccgcg ggtctggaag ttctgttcca ggggccctcc
     1621 gcgggtctgg tgccacgcgg tagtggtggt atcgaaggta ggcatatgga gctcggtacc
     1681 ctcgagggat ccgaattcaa gcttgtcgac ctgcagtcta gataggtaat ctctgcttaa
     1741 aagcacagaa tctaagatcc ctgccatttg gcggggattt ttttatttgt tttcaggaaa
     1801 taaataatcg atcgcgtaat aaaatctatt attatttttg tgaagaataa atttgggtgc
     1861 aatgagaatg cgcaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     1921 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     1981 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     2041 atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata ttttgttaaa
     2101 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2161 aatcccttat aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa
     2221 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     2281 gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg
     2341 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     2401 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     2461 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     2521 gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag atgcgtaagg
     2581 agaaaatacc gcatcaggcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2641 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2701 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     2761 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     2821 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     2881 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     2941 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     3001 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     3061 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     3121 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     3181 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     3241 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     3301 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     3361 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     3421 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     3481 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     3541 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     3601 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3661 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3721 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3781 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3841 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3901 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3961 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4021 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4081 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4141 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4201 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4261 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4321 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4381 tcctggcctt ttgctggcct tttgctcaca tagtcatgcc ccgcgcccac cggaaggagc
     4441 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4501 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4561 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4621 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4681 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4741 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4801 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     4861 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     4921 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     4981 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5041 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5101 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5161 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5221 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5281 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5341 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5401 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5461 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5521 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5581 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5641 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5701 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5761 cgccgccgc