pkk223-3大肠杆菌表达载体说明_精品文档.pdf

1、pkk223-3 编号编号 载体名称载体名称 北京华越洋生物 VECT5410 pkk223-3 pkk223-3 载体基本信息载体基本信息 载体名称载体名称:pkk223-3 质粒类型质粒类型:大肠杆菌表达载体 高拷贝高拷贝/低拷贝低拷贝:-启动子启动子:-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:4586 bp 5 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

2、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)contains the strong tac promoter upstream from themultiple cloning site and the strong rrnB ribosomal terminator downstreamfor control of protein expression(2-4).Induction:tac promoter is inducible with 1-5 mM IPTG.Promoter isvery strong;even uninduc

3、ed cells may show a low level of expression.Expression:Genes containing a ribosome-binding site and ATG can beinserted into any unique site in the MCS for expression.The ribosomebindingsite on the plasmid can be utilised to express inserts if the ATGstart codon of the insert is within 5-13 bp from t

4、he provided ribosomebindingsite(5,6).Transcription terminators:rrnB transcription terminators stabilize theplasmid by inhibiting read-through transcription initiated from the tacpromoter in the parent plasmid.Host(s):lac Iq strains;E.coli JM105 is recommended.Selectable marker(s):Plasmid confers res

5、istance to 100 g/ml ampicillin.Amplification:Recommended.Control Regions:Expression control region:tac promoter:-10:50-44;-35:72-67;Ribosome binding site:11.MCS:4552-4587.rrnB operonregion:5S rRNA region:4471-4352;rrnB T1 terminator:4348-4305;rrnB T2 terminator:4173-4146.b-lactamase gene region:Prom

6、oter:-10:4082-4077;-35:4105-4100;Start codon(ATG):4035;Stop codon(TAA):3178.Plasmid replication region:Site of replication initiation:2417-2415;Region necessary for replication:3111-2415.pkk223-3 载体序列载体序列 ORIGIN 1 TTCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACATTATA CGAGCCGATG 61 ATTAATTGTC AACAGC

7、TCAT TTCAGAATAT TTGCCAGAAC CGTTATGATG TCGGCGCAAA 121 AAACATTATC CAGAACGGGA GTGCGCCTTG AGCGACACGA ATTATGCAGT GATTTACGAC 181 CTGCACAGCC ATACCACAGC TTCCGATGGC TGCCTGACGC CAGAAGCATT GGTGCACCGT 241 GCAGTCGATA AGCTCCGGAT CCTCTACGCC GGACGCATCG TGGCCGGCAT CACCGGCGCC 301 ACAGGTGCGG TTGCTGGCGC CTATATCGCC GACA

8、TCACCG ATGGGGAAGA TCGGGCTCGC 361 CACTTCGGGC TCATGAGCGC TTGTTTCGGC GTGGGTATGG TGGCAGGCCC CGTGGCCGGG 421 GGACTGTTGG GCGCCATCTC CTTGCATGCA CCATTCCTTG CGGCGGCGGT GCTCAACGGC 481 CTCAACCTAC TACTGGGCTG CTTCCTAATG CAGGAGTCGC ATAAGGGAGA GCGTCGACCG 541 ATGCCCTTGA GAGCCTTCAA CCCAGTCAGC TCCTTCCGGT GGGCGCGGGG CA

9、TGACTATC 601 GTCGCCGCAC TTATGACTGT CTTCTTTATC ATGCAACTCG TAGGACAGGT GCCGGCAGCG 661 CTCTGGGTCA TTTTCGGCGA GGACCGCTTT CGCTGGAGCG CGACGATGAT CGGCCTGTCG 721 CTTGCGGTAT TCGGAATCTT GCACGCCCTC GCTCAAGCCT TCGTCACTGG TCCCGCCACC 781 AAACGTTTCG GCGAGAAGCA GGCCATTATC GCCGGCATGG CGGCCGACGC GCTGGGCTAC 841 GTCTTGC

10、TGG CGTTCGCGAC GCGAGGCTGG ATGGCCTTCC CCATTATGAT TCTTCTCGCT 901 TCCGGCGGCA TCGGGATGCC CGCGTTGCAG GCCATGCTGT CCAGGCAGGT AGATGACGAC 961 CATCAGGGAC AGCTTCAAGG ATCGCTCGCG GCTCTTACCA GCCTAACTTC GATCACTGGA 1021 CCGCTGATCG TCACGGCGAT TTATGCCGCC TCGGCGAGCA CATGGAACGG GTTGGCATGG 1081 ATTGTAGGCG CCGCCCTATA CCT

11、TGTCTGC CTCCCCGCGT TGCGTCGCGG TGCATGGAGC 1141 CGGGCCACCT CGACCTGAAT GGAAGCCGGC GGCACCTCGC TAACGGATTC ACCACTCCAA 1201 GAATTGGAGC CAATCAATTC TTGCGGAGAA CTGTGAATGC GCAAACCAAC CCTTGGCAGA 1261 ACATATCCAT CGCGTCCGCC ATCTCCAGCA GCCGCACGCG GCGCATCTCG GGCAGCGTTG 1321 GGTCCTGGCC ACGGGTGCGC ATGATCGTGC TCCTGTCG

12、TT GAGGACCCGG CTAGGCTGGC 1381 GGGGTTGCCT TACTGGTTAG CAGAATGAAT CACCGATACG CGAGCGAACG TGAAGCGACT 1441 GCTGCTGCAA AACGTCTGCG ACCTGAGCAA CAACATGAAT GGTCTTCGGT TTCCGTGTTT 1501 CGTAAAGTCT GGAAACGCGG AAGTCAGCGC CCTGCACCAT TATGTTCCGG ATCTGCATCG 1561 CAGGATGCTG CTGGCTACCC TGTGGAACAC CTACATCTGT ATTAACGAAG CG

13、CTGGCATT 1621 GACCCTGAGT GATTTTTCTC TGGTCCCGCC GCATCCATAC CGCCAGTTGT TTACCCTCAC 1681 AACGTTCCAG TAACCGGGCA TGTTCATCAT CAGTAACCCG TATCGTGAGC ATCCTCTCTC 1741 GTTTCATCGG TATCATTACC CCCATGAACA GAAATTCCCC CTTACACGGA GGCATCAAGT 1801 GACCAAACAG GAAAAAACCG CCCTTAACAT GGCCCGCTTT ATCAGAAGCC AGACATTAAC 1861 GC

14、TTCTGGAG AAACTCAACG AGCTGGACGC GGATGAACAG GCAGACATCT GTGAATCGCT 1921 TCACGACCAC GCTGATGAGC TTTACCGCAG CTGCCTCGCG CGTTTCGGTG ATGACGGTGA 1981 AAACCTCTGA CACATGCAGC TCCCGGAGAC GGTCACAGCT TGTCTGTAAG CGGATGCCGG 2041 GAGCAGACAA GCCCGTCAGG GCGCGTCAGC GGGTGTTGGC GGGTGTCGGG GCGCAGCCAT 2101 GACCCAGTCA CGTAGCG

15、ATA GCGGAGTGTA TACTGGCTTA ACTATGCGGC ATCAGAGCAG 2161 ATTGTACTGA GAGTGCACCA TATGCGGTGT GAAATACCGC ACAGATGCGT AAGGAGAAAA 2221 TACCGCATCA GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG 2281 CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG 2341 GATAACGCAG GAAAGAACAT GTGAGCAAAA G

16、GCCAGCAAA AGGCCAGGAA CCGTAAAAAG 2401 GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA 2461 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT 2521 GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC 2581 TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG 2641 GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC 2701 TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA 2761 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAG