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基本信息

質粒圖譜和多克隆位點信息

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載體描述

闡明蛋白質的結構和功能是后基因組研究的一個重要課題。高效率的蛋白質表達系統是研究這些課題的不可缺少的基本技術。大腸桿菌作為宿主的表達系統中被廣泛使用于生產重組蛋白。大腸桿菌表達系統具有良好的優勢,方法簡單，成本低。然而，對于某些基因，蛋白表達很困難，表達的蛋白質不溶。

為了解決這些問題，TAKARA與Masayori Inouye教授（美國新澤西醫學和牙科大學）進行聯合研究，以制定一個高效基于低溫冷休克蛋白基因的大腸桿菌表達載體。pCold DNA系列產品具有上述的優點，并提供了一個重要功能和結構的分析以及其他蛋白質研究領域的工具。

<產品概況和特點>

當培養的大腸桿菌的溫度充分降低，幾乎暫時停止生長大部分的蛋白表達減少，而此時一組蛋白質稱為“冷休克蛋白”被專門誘導表達。

冷休克表達載體，pCold I-IV, 是利用冷休克基因cspA啟動子設計高效率蛋白質表達載體。cspA的啟動子的下游處，插入了lac操縱子，以便嚴格控制表達。此外，5'非翻譯區（5'UTR），翻譯增強元件（TEE），His標簽序列，Xa因子切割位點和多克隆位點（MCS）位于在cspA啟動子的下游的。由于該產品采用了大腸桿菌的啟動子，大多數大腸桿菌菌株可被用作表達宿主。有四種類型的pCold載體，他們的原件的位置稍有不同，TEE，His標簽序列和Factor Xa酶切位點。

載體序列

LOCUS       Exported                4407 bp ds-DNA     circular SYN 16-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    .

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4407)

  AUTHORS   .

  TITLE     Direct Submission

  JOURNAL   Exported Saturday, Mar 16, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..4407

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     promoter        15..81

                     /gene="cspA"

                     /label=cspA promoter

                     /note="promoter of the E. coli cold shock protein cspA gene

                     (Mitta et al., 1997)"

     protein_bind    84..100

                     /label=lac operator

                     /bound_moiety="lac repressor encoded by 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)."

     5'UTR           122..255

                     /gene="cspA"

                     /label=cspA 5'UTR

                     /note="5'UTR of the E. coli cold shock protein cspA gene

                     (Mitta et al., 1997)"

     primer_bind     208..226

                     /label=pCold?F

     CDS             256..270

                     /codon_start=1

                     /gene="cspA"

                     /product="translation enhancing element for E. coli (Qing

                     et al., 2004)"

                     /label=TEE

                     /translation="MNHKV"

     CDS             271..288

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     CDS             289..300

                     /codon_start=1

                     /product="Factor Xa recognition and cleavage site"

                     /label=Factor Xa site

                     /translation="IEGR"

     misc_feature    301..360

                     /label=MCS

                     /note="Multi clone site"

     3'UTR           368..512

                     /gene="cspA"

                     /label=cspA 3'UTR

                     /note="3'UTR of the E. coli cold shock protein cspA gene

                     (Mitta et al., 1997)"

     primer_bind     complement(384..403)

                     /label=pCold?R

     rep_origin      complement(711..1166)

                     /direction=LEFT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow

                     indicates direction of (+) strand synthesis"

     promoter        1266..1370

                     /gene="bla"

                     /label=AmpR promoter

     CDS             1371..2231

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /label=AmpR

                     /note="confers resistance to ampicillin, carbenicillin, and

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     rep_origin      2402..2990

                     /direction=RIGHT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of

                     replication"

     CDS             complement(3162..4244)

                     /codon_start=1

                     /gene="lacI"

                     /product="lac repressor"

                     /label=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(4245..4322)

                     /gene="lacI"

                     /label=lacI promoter

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 atcatcatat cgaaggtagg

      301 catatggagc tcggtaccct cgagggatcc gaattcaagc ttgtcgacct gcagtctaga

      361 taggtaatct ctgcttaaaa gcacagaatc taagatccct gccatttggc ggggattttt

      421 ttatttgttt tcaggaaata aataatcgat cgcgtaataa aatctattat tatttttgtg

      481 aagaataaat ttgggtgcaa tgagaatgcg caggcccttt cgtctcgcgc gtttcggtga

      541 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc

      601 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg

      661 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata aaattgtaaa

      721 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca

      781 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga tagggttgag

      841 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg

      901 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt

      961 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag

     1021 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc

     1081 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc

     1141 gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg gtgtgaaata

     1201 ccgcacagat gcgtaaggag aaaataccgc atcaggcgtc aggtggcact tttcggggaa

     1261 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca

     1321 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc

     1381 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc

     1441 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt

     1501 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt

     1561 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg

     1621 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact

     1681 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg

     1741 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga

     1801 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg

     1861 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa

     1921 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac

     1981 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc

     2041 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca

     2101 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga

     2161 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta

     2221 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc

     2281 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc

     2341 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt

     2401 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac

     2461 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct

     2521 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact

     2581 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg

     2641 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata

     2701 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga

     2761 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag

     2821 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg

     2881 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac

     2941 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca

     3001 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacata gtcatgcccc

     3061 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg

     3121 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt

     3181 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt

     3241 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg

     3301 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc

     3361 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta

     3421 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg

     3481 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc

     3541 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt

     3601 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga

     3661 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg

     3721 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg

     3781 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca

     3841 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg

     3901 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc

     3961 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc

     4021 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt

     4081 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc

     4141 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag

     4201 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat

     4261 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg

     4321 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag

     4381 gttgaggccg ttgagcaccg ccgccgc

//