质粒名称: | pcDNA3.1(+)/Puro/FA/GFP-N--Grb2-2 |
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出品公司: | addgene |
目录编号: | 112837 |
存储实验室: | Tomasz Prószyński |
载体骨架基本信息 |
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载体名称: | pcDNA3.1(+)/Puro |
空载体大小: | 5500 bp |
完整质粒大小: | 6363bp |
载体类型: | 哺乳细胞表达 |
筛选标记: | Puromycin |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | Grb2 |
物种来源: | M. musculus (mouse) |
基因突变: | SH2 + SH3 (C-term) domains of Grb2 |
标签/融合蛋白: | GFP (N terminal on insert) |
克隆方法: | 限制性内切酶 |
5’克隆位点: | 未知(未破坏) |
3’克隆位点: | 未知(未破坏) |
文献引用方法 |
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材料和方法部分: | pcDNA3.1(+)/Puro/FA/GFP-N--Grb2-2 was a gift from Tomasz Prószyński (Addgene plasmid # 112837 ; http://n2t.net/addgene:112837 ; RRID:Addgene_112837) |
参考文献部分: | Alpha-Dystrobrevin-1 recruits Grb2 and alpha-catulin to organize neurotransmitter receptors at the neuromuscular junction. Gingras J, Gawor M, Bernadzki KM, Grady RM, Hallock P, Glass DJ, Sanes JR, Proszynski TJ. J Cell Sci. 2016 Mar 1;129(5):898-911. doi: 10.1242/jcs.181180. Epub 2016 Jan 14. 10.1242/jcs.181180 PubMed 26769899 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pcDNA3.1(+)/Puro/FA/GFP-N--Grb2-2 6363 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION transient/stable overexpression. ACCESSION . VERSION . KEYWORDS pcDNA3.1(+)/Puro/FA/GFP-N--Grb2-2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6363) AUTHORS Gingras J, Gawor M, Bernadzki KM, Grady RM, Hallock P, Glass DJ, Sanes JR, Proszynski TJ TITLE Alpha-Dystrobrevin-1 recruits Grb2 and alpha-catulin to organize neurotransmitter receptors at the neuromuscular junction. JOURNAL J Cell Sci. 2016 Mar 1;129(5):898-911. doi: 10.1242/jcs.181180. Epub 2016 Jan 14. PUBMED 26769899 REFERENCE 2 (bases 1 to 6363) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..6363 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 629..648 /label=T7 /note="T7 promoter, forward primer" promoter 629..647 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 701..1417 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 701..1417 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(746..767) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1007..1026) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1354..1375 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1975..1992) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1981..2205 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2251..2679 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2338..2357) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2548..2569 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2688..2708) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2693..3022 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2873..3008 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2935..2954 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3043..3642 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(3043..3062) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3539..3559 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 3802..3923 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3839..3858) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3893..3912 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3972..3988) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3972..3988) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3985..4007) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3996..4012 /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)." promoter complement(4020..4050) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4065..4086 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4203..4220) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4374..4962) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4454..4473) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5133..5993) /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 5756..5775 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5994..6098) /gene="bla" /label=AmpR promoter primer_bind complement(6173..6192) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg 661 gctagcgttt aaacttaagc ttggtaccga gctcggatcc atggtgagca agggcgagga 721 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 781 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt 841 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 901 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc 961 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta 1021 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 1081 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa 1141 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa 1201 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 1261 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc 1321 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 1381 cgccgggatc actctcggca tggacgagct gtacaagggc cggcctgtgg tggaattccc 1441 gtggtttttt ggcaaaatcc ccagagccaa ggcagaagaa atgctcagca aacagcggca 1501 tgacggggcc ttcctgatcc gagagagcga gagcgctcct ggggacttct ccctgtccgt 1561 caagtttgga aatgatgtgc agcacttcaa ggtgctccgc gacggagccg ggaagtattt 1621 cctgtgggtg gtgaagttta attctttgaa tgagctggta gattaccaca gatcaacatc 1681 cgtgtccagg aaccagcaga tattcttacg ggacatagaa cagatgccac agcagccaac 1741 ctacgtccag gcgctctttg actttgaccc ccaggaggat ggcgagctgg gctttcgcag 1801 aggagacttc attcatgtca tggataactc agatcccaat tggtggaaag gggcctgcca 1861 cgggcagacc ggcatgtttc cccgcaatta tgtcaccccc gtgaaccgga acgtcgatat 1921 ccagcacagt ggcgcgcctc gagtctagag ggcccgttta aacccgctga tcagcctcga 1981 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 2041 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 2101 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 2161 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 2221 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg 2281 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 2341 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 2401 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 2461 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 2521 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 2581 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 2641 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca 2701 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 2761 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 2821 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc 2881 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 2941 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt 3001 tggaggccta ggcttttgca aaaagctccc ggggccgcca ccatgaccga gtacaagccc 3061 acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg 3121 ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc 3181 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc 3241 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg 3301 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag 3361 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc 3421 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc 3481 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc 3541 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa 3601 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gattcgaaat gaccgaccaa 3661 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg 3721 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 3781 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc 3841 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 3901 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg 3961 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 4021 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 4081 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 4141 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 4201 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 4261 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 4321 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 4381 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 4441 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 4501 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 4561 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4621 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 4681 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 4741 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 4801 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 4861 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 4921 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 4981 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 5041 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 5101 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 5161 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 5221 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 5281 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 5341 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 5401 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 5461 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 5521 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 5581 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 5641 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 5701 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 5761 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 5821 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 5881 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 5941 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 6001 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 6061 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 6121 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct 6181 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 6241 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 6301 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 6361 gtt //