九种载体的多克隆位点及质粒图谱文档格式.docx
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九种载体的多克隆位点及质粒图谱文档格式.docx
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PCR引物设计时BamHI和EcoRI位点的5端保护碱基有2至3个GC即可,而XbaI、NheI和SpeI位点5端保护碱基须在5个左右才容易被酶切割。
(一)pLLp-OmpA
Size:
7.5Kb
多克隆位点:
PlpplacoperatorRBS
CGAGGCGCAAAAAATGAAAAAGACAGCTATCGCG
MetLysLysThrAlaIleAla
OmpAsignal
ATTGCAGTGGCACTGGCAGGTTTCGCTACCGTCGCTCAGGCTGGATCCGCA
IleAlaValAlaLeuAlaGlyPheAlaThrValAlaGlnAlaBamHI
GAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTT
EcoRINheI6His-tagHindIII
BamHIEcoRINheI6His-stopHindIII
PlpplacoperatorRBS—ATG—OmpAsignal—MCS—
基因克隆表达功能区序列如下,经DNA双向测序验证ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagataacgaggcgcaaaaaatgaaaaagacagctatcgcgattgcagtggcactggcaggtttcgctaccgtcgctcaggctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta
载体其它部分序列见GenebankLocusASAK3121(plasmidpIN-III-ompA1)
(二)pLLp-STII
7.5kb
多克隆住点:
PlpplacoperatorRBS
GAAGGAGATATACATATGAAAAAGAACATCGCATTCCTC
MetLysLysAsnIleAlaPheLeu
STII signal
CTGGCATCTATGTTTGTTTTCTCTATCGCTACCAACGCTTACGCTGGATCC
LeuAlaSerMetPheValPheSerIleAlaThrAsnAlaTyrAlaBammHI
GCAGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTT
NheI6His-tagHindIII
PlpplacoperatorATG—STIIsignalpeptide—MCS—
基因克隆表达功能区序列如下,经DNA双向测序验证
ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagaaataattttgtttaactttaagaaggagatatacatatgaaaaagaacatcgcattcctcctggcatctatgtttgttttctctatcgctaccaacgcttacgctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta
STIIsignalpeptide见GenebankLocusHSTI_ECOLI(p22542)
(三)pMBP-p
Vectorsize:
6.7kb
Ptaclacoperator
分泌型maltosebindingproteinAACAACAACAACAACAACAACAAT
Thrombincutsite
AACAACCTGGGTATCGATACTACTGAGAACTATCCGCGTGGATCCGCAGAA
AsnTyrProArgBamHIEcoRI
TTCAGCGCTAGCCACCATCATCATCATCATTAAGCTT
NheI6His-tagHindIII
Ptaclacoperator分泌型MBP—thrombincutsiteMCS
Gccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagctt
HindIII
载体其它部分序列见pMal-p2
Maltosebindingprotein(E.coli)编码氨基酸序列见Genebank
MBPMW=42KD
(四)pMBP-c
6.6kb
胞内maltosebindingproteinAACAACAACAACAACAACAACAAT
Ptaclacoperator胞内MBPthrombincutsite—MCS
载体其它部分序列见pMal-c2
(五)pET-GST
vectorsize:
3.7kb
P3Ccutsite
PT7RBS—ATG—GST—CTCGAAGTTCTGTTTCAGGGTCCAGCAGGA
LeuGluValLeuPheGlnGlyProBamHI
TCCGCAGAATTCAGCGCTAGCCACCATCATCATCATTAAGCTT
EcoRINheI6His-tagHindII
BamHIEcoRINheI6HisstopHindIII
PT7GSTP3ccutsite—MCS—
XbaI
tctagatcacacaggaaacagtattcatgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggacagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatcatcattaagctt
HindIII
载体其它部分序列见pUC18(不含pUC18多克隆位点)
GlutathioneS-transferase编码氨基酸序列如下(含多克隆位点):
MSPILGYMKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDSMPDSLEVLFQGPAGSAEFSASHHHHHH
248AMINOACIDS
MW28.7KD
(六)pET-Trx
3.3kb
PT7RBS—TRX(his-patch—CTCGAAGTTCTGTTTCAGGGTCCAGCAGGA
TCCGCAGAATTCAGCGCTAGCTAACATCATCATCATTAAGCTT
EcoRINheIstopHindIII
BamHIEcoRINheIstopHindIII
PT7p3ccutsite—MCS—
tctagaaataattttgtttaactttaagaaggagatatacatatgtctgataaaattattcatctgactgatgattcttttgatactgatgtacttaaggcagatggtgcaatcctggttgatttctgggcacactggtgcggtccgtgcaaaatgatcgctccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcacaacccgggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggctggctctagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagctaacatcatcatcatcattaagctt
Thioredoxinmut(His-patch)(E.coli)编码氨基酸序列如下(含多克隆位点):
MSDKIIHLTDDSFDTDVLKADGAILVDFWAHWCGPCKMIAPILDEIADEYQGKLTVAKLNIDHNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSSMPDSLEVLFQGPAGSAEFSAS
133AMINOACIDS
MW14KD
3个His形成立体His-patch
(七)pET-His
2.9kb
thrombincutsite
PT7RBS—ATGHisCTGGTGCCGCGCGGATCCGCAGAATTCAGCGCT
LeuValProArgBamHIEcoRI
AGCTAACATCATCATCATCATTAAGCTT
NheIHindIII
PT7ATG-6HisThrombincutsite—MCS—
tctagaaataattttgtttaactttaagaaggagatataccatgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggatccgcagaattcagcgctagctaacatcatcatcatcattaagctt
载体其它部分序列见pUC18(不含pUC18多克隆位点)
(八)PTRC-CKS
5.2kb
PTrcRBS—ATG—CKSmut—CTCGAAGTTCTGTTTCAGGGTCCAGCAGGA
TCCGCAGAATTCAGCGCTAGCCACCATCATCATCATCATTAAGCTT
EcoRINheI6His-tagHindIII
BamHIEcoRINheI6His-stopHindIII
PTrc—RBS—ATG—CKSmut—p3Ccutsite—MCS
tctagaaataattttgtttaactttaagaaggagatatacatatgagttttgtggtcattattcccgcgcgctacgcgtccacgcgtctgcccggtaaaccattggttgatattaacggcaaacccatgattgttcatgttcttgaacgcgcgcgtgaatcaggtgccgagcgcatcatcgtggcaaccgatcatgaggatgttgcccgcgccgttgaagccgctggcggtgaagtatgtatgacgcgcgccgatcatcagtcaggaacagaacgtctggcggaagttgtcgaaaaatgcgcattcagcgacgacacggtgatcgttaatgtgcagggtgatgaaccgatgatccctgcgacaatcatccgtcaggttgctgataacctcgctcagcgtcaggtgggtatggcgactctggcggtgccaatccacaatgcggaagaagcgtttaacccgaatgcggtgaaagtggttctcgacgctgaagggtatgcactgtacttctctcgcgccaccattccttgggatcgtgatcgttttgcagaaggccttgaaaccgttggcgataacttcctgcgtcatcttggtatttatggctaccgtgcaggctttatccgtcgttacgtcaactggcagccaagtccgttagaacacatcgaaacgttagagcagcttcgtgttctgtggtacggcgaaaaaatccatgttgctgttgctcaggaagttcctggcacaggtgtggatacccctgaggacggtagcatgccagactctctcgaagttctgtttcagggtccagcaggatccgcagaattcagcgctagccaccatcatcatcatcattaagctt
载体其它部分序列见pTrcHisB(不含pTrcHisB多克隆位点)
CKSmut(E.coliCTP:
CMP-3-deoxy-D-manno-octulosonatecytidylyltransferaseorCMP-KDOsynthetase)编码氨基酸序列如下(含多克隆位点):
MSFVVIIPARYASTRLPGKPLVDINGKPMIVHVLERARESGAERIIVATDHEDVARAVEAAGGEVCMTRADHQSGTERLAEVVEKCAFSDDTVIVNVQGDEPMIPATIIRQVADNLAQRQVGMATLAVPIHNAEEAFNPNAVKVVLDAEGYALYFSRATIPWDRDRFAEGLETVGDNFLRHLGIYGYRAGFIRRYVNWQPSPLEHIETLEQLRVLWYGEKIHVAVAQEVPGTGVDTPEDGSMPDSLEVLFQGPAGSAEFSASHHHHHH
268AminoAcidsMolecularWeight29KD
(九)PET-DsbA
3.6kb
多克隆位点t
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