质粒名称: | pBSFI-myr-akt-T308A/S473A |
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出品公司: | 爱迪基因 |
目录编号: | 49191 |
存储实验室: | Peter Vogt |
载体骨架基本信息 |
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载体名称: | pBSFI |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | 3000 bp |
完整质粒大小: | 4500 bp |
载体修饰: | pBSFI基于pBluescript SK,在SacI和KpnI之间插入了不同的MCS |
载体类型: | Adaptor Plasmid |
筛选标记: | -- |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | Akt1,Akt |
物种来源: | M. musculus (mouse) |
插入大小: | 1500 bp |
基因突变: | T308A; S473A |
GenBank编号 | NM_009652.3 |
启动子 | T7 |
标签/融合蛋白: |
myristolation signal(插入件上的N端子)
HA tag (插接件上的C端子)
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克隆方法: | 限制性内切酶 |
5' 克隆位点: | BamHI(克隆过程中销毁) |
3' 克隆位点: | EcoRI(未销毁) |
5' 测序引物 | T3 |
3' 测序引物 | T7 |
文献引用方法 |
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材料和方法部分: | pBSFI-myr-akt-T308A/S473A was a gift from Peter Vogt (Addgene plasmid # 49191 ; http://n2t.net/addgene:49191 ; RRID:Addgene_49191) |
参考文献部分: | The akt kinase: molecular determinants of oncogenicity. Aoki M, Batista O, Bellacosa A, Tsichlis P, Vogt PK. Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14950-5. 10.1073/pnas.95.25.14950 PubMed 9843996 |
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载体基本信息
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载体序列
LOCUS pBSFI-myr-akt-T308A/S473A 4474 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION cDNA of Akt1 with myristyolation signal and point mutation from T to A at position 308 and S to A at position 473 in cloning vector. ACCESSION . VERSION . KEYWORDS pBSFI-myr-akt-T308A/S473A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4474) AUTHORS Aoki M, Batista O, Bellacosa A, Tsichlis P, Vogt PK TITLE The akt kinase: molecular determinants of oncogenicity. JOURNAL Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14950-5. PUBMED 9843996 REFERENCE 2 (bases 1 to 4474) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4474 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1536..1562 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(1631..1650) /label=T7 /note="T7 promoter, forward primer" promoter complement(1632..1650) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1657..1674) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1657..1673) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1666..1688) /label=M13/pUC Forward /note="In lacZ gene" rep_origin complement(1814..2269) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1951..1972) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2163..2182 /label=F1ori-R /note="F1 origin, reverse primer" promoter 2296..2400 /gene="bla" /label=AmpR promoter CDS 2401..3261 /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" primer_bind complement(2619..2638) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3432..4020 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3921..3940 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4174..4191 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4308..4329 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4344..4374 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4382..4398 /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)." primer_bind 4387..4409 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4406..4422 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4406..4422 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 4443..4461 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 agctggccat tacggccgcg gccgcctcta gaggatctgg taccacgcgt atcgataagc 61 ttatggggag cagcaagagc aagcccaagt ctagaatgaa cgacgtagcc attgtgaagg 121 agggctggct gcacaaacga ggggaatata ttaaaacctg gcggccacgc tacttcctcc 181 tcaagaacga tggcaccttt attggctaca aggaacggcc tcaggatgtg gatcagcgag 241 agtccccact caacaacttc tcagtggcac aatgccagct gatgaagaca gagcggccaa 301 ggcccaacac ctttatcatc cgctgcctgc agtggaccac agtcattgag cgcaccttcc 361 atgtggaaac gcctgaggag cgggaagaat gggccaccgc cattcagact gtggccgatg 421 gactcaagag gcaggaagaa gagacgatgg acttccgatc aggctcaccc agtgacaact 481 caggggctga agagatggag gtgtccctgg ccaagcccaa gcaccgtgtg accatgaacg 541 agtttgagta cctgaaacta ctgggcaagg gcacctttgg gaaagtgatt ctggtgaaag 601 agaaggccac aggccgctac tatgccatga agatcctcaa gaaggaggtc atcgtcgcca 661 aggatgaggt tgcccacacg cttactgaga accgtgtcct gcagaactct aggcatccct 721 tccttacggc cctcaagtac tcattccaga cccacgaccg cctctgcttt gtcatggagt 781 atgccaacgg gggcgagctc ttcttccacc tgtctcgaga gcgcgtgttc tccgaggacc 841 gggcccgctt ctatggtgcg gagattgtgt ctgccctgga ctacttgcac tccgagaaga 901 acgtggtgta ccgggacctg aagctggaga acctcatgct ggacaaggac gggcacatca 961 agataacgga cttcgggctg tgcaaggagg ggatcaagga tggtgccact atgaaggcat 1021 tctgcggaac gccggagtac ctggcccctg aggtgctgga ggacaacgac tacggccgtg 1081 cagtggactg gtgggggctg ggcgtggtca tgtatgagat gatgtgtggc cgcctgccct 1141 tctacaacca ggaccacgag aagctgttcg agctgatcct catggaggag atccgcttcc 1201 cgcgcacact cggccctgag gccaagtccc tgctctccgg gctgctcaag aaggacccta 1261 cacagaggct cggtgggggc tctgaggatg ccaaggagat catgcagcac cggttctttg 1321 ccaacatcgt gtggcaggat gtgtatgaga agaagctgag cccacctttc aagccccagg 1381 tcacctctga gactgacacc aggtatttcg atgaggagtt cacagctcag atgatcacca 1441 tcacgccgcc tgatcaagat gacagcatgg agtgtgtgga cagtgagcgg aggccgcact 1501 tcccccagtt cgcctactca gccagtggca cagcctaccc atacgatgtt ccagattacg 1561 cttctagatg aattcacgcg taagcttatc gatgtcgacg ggcccggccg cctcggccgt 1621 acccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg 1681 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc 1741 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct 1801 gaatggcgaa tggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt 1861 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa 1921 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag 1981 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt 2041 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 2101 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 2161 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 2221 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact 2281 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 2341 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 2401 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 2461 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 2521 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 2581 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 2641 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 2701 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 2761 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 2821 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 2881 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 2941 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 3001 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 3061 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 3121 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 3181 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 3241 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 3301 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 3361 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 3421 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 3481 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 3541 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 3601 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 3661 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 3721 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 3781 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 3841 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 3901 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 3961 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 4021 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 4081 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 4141 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 4201 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg 4261 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag 4321 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga 4381 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc 4441 gaaattaacc ctcactaaag ggaacaaaag ctgg //