pkk223-3大肠杆菌表达载体说明_精品文档资料下载.pdf

pkk223-3大肠杆菌表达载体说明_精品文档资料下载.pdf

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-克隆方法克隆方法:

多克隆位点，限制性内切酶载体大小载体大小:

4586bp5测序引物及序列测序引物及序列:

-3测序引物及序列测序引物及序列:

-载体标签载体标签:

-载体抗性载体抗性:

Ampicillin筛选标记筛选标记:

-备注备注:

-稳定性稳定性:

-组成型组成型:

-病毒病毒/非病毒非病毒:

非病毒pkk223-3载体质粒图谱和多克隆位点信息载体质粒图谱和多克隆位点信息pkk223-3载体简介载体简介pKK223-3

（1）containsthestrongtacpromoterupstreamfromthemultiplecloningsiteandthestrongrrnBribosomalterminatordownstreamforcontrolofproteinexpression（2-4）.Induction:

tacpromoterisinduciblewith1-5mMIPTG.Promoterisverystrong;

evenuninducedcellsmayshowalowlevelofexpression.Expression:

Genescontainingaribosome-bindingsiteandATGcanbeinsertedintoanyuniquesiteintheMCSforexpression.TheribosomebindingsiteontheplasmidcanbeutilisedtoexpressinsertsiftheATGstartcodonoftheinsertiswithin5-13bpfromtheprovidedribosomebindingsite（5,6）.Transcriptionterminators:

rrnBtranscriptionterminatorsstabilizetheplasmidbyinhibitingread-throughtranscriptioninitiatedfromthetacpromoterintheparentplasmid.Host（s）:

lacIqstrains;

E.coliJM105isrecommended.Selectablemarker（s）:

Plasmidconfersresistanceto100g/mlampicillin.Amplification:

Recommended.ControlRegions:

Expressioncontrolregion:

tacpromoter:

-10:

50-44;

-35:

72-67;

Ribosomebindingsite:

11.MCS:

4552-4587.rrnBoperonregion:

5SrRNAregion:

4471-4352;

rrnBT1terminator:

4348-4305;

rrnBT2terminator:

4173-4146.b-lactamasegeneregion:

Promoter:

4082-4077;

4105-4100;

Startcodon（ATG）:

4035;

Stopcodon（TAA）:

3178.Plasmidreplicationregion:

Siteofreplicationinitiation:

2417-2415;

Regionnecessaryforreplication:

3111-2415.pkk223-3载体序列载体序列ORIGIN1TTCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGATG61ATTAATTGTCAACAGCTCATTTCAGAATATTTGCCAGAACCGTTATGATGTCGGCGCAAA121AAACATTATCCAGAACGGGAGTGCGCCTTGAGCGACACGAATTATGCAGTGATTTACGAC181CTGCACAGCCATACCACAGCTTCCGATGGCTGCCTGACGCCAGAAGCATTGGTGCACCGT241GCAGTCGATAAGCTCCGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCC301ACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGC361CACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGG421GGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGC481CTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCG541ATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATC601GTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCG661CTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCG721CTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACC781AAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTAC841GTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCT901TCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGAC961CATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCACTGGA1021CCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGG1081ATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGC1141CGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAA1201GAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGA1261ACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTG1321GGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGC1381GGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACT1441GCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTT1501CGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCG1561CAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATT1621GACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCAC1681AACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTC1741GTTTCATCGGTATCATTACCCCCATGAACAGAAATTCCCCCTTACACGGAGGCATCAAGT1801GACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAAC1861GCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCT1921TCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGA1981AAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG2041GAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCAT2101GACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAG2161ATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAA2221TACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGG2281CTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGG2341GATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAG2401GCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA2461CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT2521GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC2581TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG2641GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGC2701TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCA2761CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG