九种载体的多克隆位点及质粒图谱
（pLLP-OmpA、pLLP-STII、pMBP-P、pMBP-C、pTrc-CKS、pET-GST、pET-Trx、pET-His、pET-DsbA）
这九种载体的多克隆位点均为
GGA TCC GCA GAA TTC AGC GCT AGC CAC（TAA） CAT CAT BamHI EcoRI NheI His His His CAT CAT CAT TAA GCTT His His His HindIII
我们建议您扩增基因5 ?端选BamHI或BglII，3?端选NheI或XbaI或SpeI，同时3?端不要带终止密码子，注意与GGA TCC（Gly Ser）表达框架正确，载体用NheI—BamHI双酶切。BamHI和BglII为同裂酶，NheI、XbaI和SpeI为同裂酶。
如您目的基因无法满足上述要求，如同时含有BamHI和BglII，5?端可选择EcoRI，但须注意与表达框架GAA TTC（Glu Phe）对齐。如您目的基因同时含有XbaI、NheI和SpeI，3?端可选择EcoRI。
如果您的目的基因已带有终止密码子，同样可以进行克隆表达，只是注意有些表达载体是在C端带6个His。
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：
Plpp lac operator RBS
CGAGGCGCAAAAA ATG AAA AAG ACA GCT ATC GCG Met Lys Lys Thr Ala Ile Ala OmpA signal
ATT GCA GTG GCA CTG GCA GGT TTC GCT ACC GTC GCT CAG GCT GGA TCC GCA Ile Ala Val Ala Leu Ala Gly Phe Ala Thr Val Ala Gln Ala BamHI GAA TTC AGC GCT AGC CAC CAT CAT CAT CAT CAT TAAGCTT EcoRI NheI 6His-tag HindIII
BamHI EcoRI NheI 6His- stop HindIII
Plpp lac operator RBS—ATG—OmpA signal—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Genebank Locus ASAK3121 (plasmid pIN-III-ompA1) （二）pLLp-STII
Size: 7.5kb 多克隆住点：
Plpp lac operator RBS
GAAGGAGATATACAT ATG AAA AAG AAC ATC GCA TTC CTC Met Lys Lys Asn Ile Ala Phe Leu STII signal
CTG GCA TCT ATG TTT GTT TTC TCT ATC GCT ACC AAC GCT TAC GCT GGA TCC
Leu Ala Ser Met Phe Val Phe Ser Ile Ala Thr Asn Ala Tyr Ala BammHI GCA GAA TTC AGC GCT AGC CAC CAT CAT CAT CAT CAT TAA GCTT NheI 6His-tag HindIII
BamHI EcoRI NheI 6His- stop HindIII
Plpp lac operator ATG—STII signal peptide—MCS—
基因克隆表达功能区序列如下，经DNA双向测序验证
ctcactttatctaagatgaatccgatggaagcatcctgttttctctcaatttttttatctaaaacccagcgttcgatgcttctttgagcgaacgatcaaaaataagtgccttcccatcaaaaaaatattctcaacataaaaaactttgtgtaatacttgtaacgctacatggagattaactcaatctagctagagaggctttacactttatgcttccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggaactctagaaataattttgtttaactttaagaaggagatatacatatgaaaaagaacatcgcattcctcctggcatctatgtttgttttctctatcgctaccaacgcttacgctggatccgcagaattcagcgctagccaccatcatcatcatcattaagcttagatccggctgagcaacgacgtgaacgcaatgcgttccgacgttcaggctgctaaagatgacgcagctcgtgctaaccagcgtctggacaacatggctactaaataccgcaagtaatagtacctgtgaagtgaaaaatggcgcacattgtgcgacattttttttgtctgccgtttaccgctactgcgtcacgcgtaacatattcccttgctctggttcaccattctgcgctgactctactgaaggcgcattgctggctgcgggagttgctccactgctcaccgaaaccggataccctgcccgacgatacaacgctttatcgacta
载体其它部分序列见Genebank Locus ASAK3121 (plasmid pIN-III-ompA1) STII signal peptide见Genebank Locus HSTI_ECOLI (p22542)
(三)pMBP-p
Vector size: 6.7kb
Ptac lac operator 分泌型maltose binding protein AAC AAC AAC AAC AAC AAC AAC AAT Thrombin cut site
AAC AAC CTG GGT ATC GAT ACT ACT GAG AAC TAT CCG CGT GGA TCC GCA GAA Asn Tyr Pro Arg BamHI EcoRI
TTC AGC GCT AGC CAC CAT CAT CAT CAT CAT TAA GCTT NheI 6His-tag HindIII
BamHI EcoRI NheI 6His- stop HindIII
Ptac lac operator 分泌型 MBP—thrombin cut site MCS
基因克隆表达功能区序列如下，经DNA双向测序验证
Gccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagctt HindIII
载体其它部分序列见pMal-p2
Maltose binding protein(E.coli)编码氨基酸序列见Genebank MBP MW=42KD
(四)pMBP-c
Vector size: 6.6kb Ptac lac operator 胞内maltose binding protein AAC AAC AAC AAC AAC AAC AAC AAT Thrombin cut site
AAC AAC CTG GGT ATC GAT ACT ACT GAG AAC TAT CCG CGT GGA TCC GCA GAA Asn Tyr Pro Arg BamHI EcoRI
TTC AGC GCT AGC CAC CAT CAT CAT CAT CAT TAA GCTT NheI 6His-tag HindIII
BamHI EcoRI NheI 6His- stop HindIII
Ptac lac operator 胞内 MBP thrombin cut site—MCS
基因克隆表达功能区序列如下，经DNA双向测序验证
Gccaccatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactaattcgagctcgaacaacaacaacaataacaataacaacaacctgggtatcgatactactgagaactatccgcgtggatccgcagaattcagcgctagccaccatcatcatcatcattaagctt HindIII
载体其它部分序列见pMal-c2
Maltose binding protein(E.coli)编码氨基酸序列见Genebank MBP MW=42KD
（五）pET-GST vector size: 3.7kb 多克隆位点：
P3C cut site
PT7 RBS — ATG—GST —CTC GAA GTT CTG TTT CAG GGT CCA GCA GGA Leu Glu Val Leu Phe Gln Gly Pro BamHI TCC GCA GAA TTC AGC GCT AGC CAC CAT CAT CAT CAT TAA GCTT EcoRI NheI 6His-tag HindII