| 质粒名称: | pT3-Neo-EF1a-GFP |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 69134 |
| 存储实验室: | Martin Bonamino |
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载体骨架基本信息 |
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| 载体名称: | pT3 |
| 完整质粒大小: | 7159 bp |
| 载体类型: | 哺乳细胞表达、Sleeping Beauty transposon |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
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插入基因信息 |
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| 启动子: | U3MSCV |
| 克隆方法: | 限制性内切酶 |
| 5’测序引物 | ACTAACCAATCAGTTCGCTTCTC |
| 3’测序引物 | TAGAAGGCACAGTCGAGG |
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文献引用方法 |
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| 材料和方法部分: | pT3-Neo-EF1a-GFP was a gift from Martin Bonamino (Addgene plasmid # 69134 ; http://n2t.net/addgene:69134 ; RRID:Addgene_69134) |
| 参考文献部分: | An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells. Chicaybam L, Barcelos C, Peixoto B, Carneiro M, Limia CG, Redondo P, Lira C, Paraguassu-Braga F, Vasconcelos ZF, Barros L, Bonamino MH. Front Bioeng Biotechnol. 2017 Jan 23;4:99. doi: 10.3389/fbioe.2016.00099. eCollection 2016. 10.3389/fbioe.2016.00099 PubMed 28168187 |
买家导航
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 pT3-Neo-EF1a-GFP 7159 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Sleeping Beaquty (SB) vector encoding G418 resistance and GFP from two separate promoters. ACCESSION . VERSION . KEYWORDS pT3-Neo-EF1a-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7159) AUTHORS Chicaybam L, Barcelos C, Peixoto B, Carneiro M, Limia CG, Redondo P, Lira C, Paraguassu-Braga F, Vasconcelos ZF, Barros L, Bonamino MH TITLE An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells. JOURNAL Front Bioeng Biotechnol. 2017 Jan 23;4:99. doi: 10.3389/fbioe.2016.00099. eCollection 2016. PUBMED 28168187 REFERENCE 2 (bases 1 to 7159) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..7159 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 64..1242 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 295..1233 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1190..1210 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 1319..1328 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1325..2041 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1325..2041 /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(1370..1391) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1631..1650) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1978..1999 /label=EGFP-C /note="EGFP, forward primer" regulatory 2520..2529 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter complement(3355..3373) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3356..3373) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(3393..3410) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3399..3623 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(4062..4078) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4062..4078) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4075..4097) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4086..4102 /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(4110..4140) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4155..4176 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4293..4310) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4464..5052) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4544..4563) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5223..6083) /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 5846..5865 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6084..6188) /gene="bla" /label=AmpR promoter primer_bind 6256..6274 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(6312..6334) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 6434..6453 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6647..6669 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 6661..6678 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 6662..6678 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7060..7080) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" ORIGIN 1 ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa aggagtggga 61 attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg 121 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa 181 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt 241 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt 301 gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga 361 attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg 421 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc 481 tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct 541 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc 601 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg 661 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg 721 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg 781 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca 841 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg 901 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt 961 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc 1021 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg 1081 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg 1141 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag 1201 acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggaattct gcagtcgatc 1261 gacggtaccg cgggcccggg atccaggcct aagcttacgc gtcctagcgc taccggtcgc 1321 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 1381 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 1441 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 1501 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat 1561 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 1621 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 1681 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 1741 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 1801 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 1861 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 1921 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 1981 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 2041 gtccggactc agatctcgac tagctagtag ctagctagct agtcgagctc aagcttcgat 2101 actagtttaa ttaagatctc gagggaatga aagaccccac ctgtaggttt ggcaagctag 2161 cttaagtaac gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag 2221 atcaaggtta ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc 2281 agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag 2341 cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt 2401 gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc 2461 cgagctcaat aaaagagccc acaacccctc actcggcgcg ccagtccgga tccaccggtg 2521 ccaccatggc cgaacaggat ggactgcacg caggctcccc cgccgcttgg gtggaaagac 2581 tgtttggata cgactgggct cagcagacaa tcggatgctc tgacgcagcc gtgttccggc 2641 tgagtgctca ggggcgcccc gtgctgtttg tcaagacaga cctgtcagga gcactgaacg 2701 agctgcagga tgaagcagcc cgcctgagct ggctggctac cacaggagtg ccttgtgctg 2761 cagtcctgga cgtggtcact gaggcaggcc gagattggct gctgctgggg gaagtgccag 2821 gacaggacct gctgagctcc cacctggcac cagctgagaa ggtctccatc atggcagatg 2881 ccatgcggag actgcatact ctggaccccg ccacctgccc tttcgatcac caggctaaac 2941 atcgcattga gagggctcgc acccgaatgg aagcaggcct ggtggaccag gacgatctgg 3001 atgaggaaca ccaggggctg gctcctgcag agctgtttgc acggctgaaa gccagaatgc 3061 cagacggaga agatctggtg gtcacccatg gcgacgcttg cctgcccaac atcatggtgg 3121 agaatggcag attcagtggg tttattgatt gtggacgact gggcgtcgcc gacagatacc 3181 aggatatcgc cctggctaca agagacattg cagaggaact gggcggggaa tgggccgata 3241 ggttcctggt gctgtatggc atcgccgcac ctgattcaca gaggattgct ttttacagac 3301 tgctggatga gtttttctaa gcggccgctc gagcatgcat ctagagggcc ctattctata 3361 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 3421 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 3481 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 3541 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 3601 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctgca ttaatggatc 3661 cccttgaaat acatccacag gtacacctcc aattgactca aatgatgtca attagtctat 3721 cagaagcttc taaagccatg acatcatttt ctggaatttt ccaagctgtt taaaggcaca 3781 gtcaacttag tgtatgtaaa cttctgaccc actggaattg tgatacagtg aattataagt 3841 gaaataatct gtctgtaaac aattgttgga aaaatgactt gtgtcatgca caaagtagat 3901 gtcctaactg acttgccaaa actattgttt gttaacaaga aatttgtgga gtagttgaaa 3961 aacgagtttt aatgactcca acttaagtgt atgtaaactt ccgacttcaa ctgtataggt 4021 ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg 4081 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 4141 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 4201 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 4261 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 4321 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 4381 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 4441 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 4501 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4561 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 4621 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 4681 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 4741 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 4801 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 4861 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc 4921 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 4981 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 5041 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 5101 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 5161 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 5221 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 5281 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 5341 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 5401 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 5461 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 5521 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 5581 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 5641 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 5701 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 5761 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 5821 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 5881 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 5941 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 6001 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 6061 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 6121 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 6181 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg 6241 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat 6301 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg 6361 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 6421 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa 6481 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg 6541 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 6601 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 6661 tgtaaaacga cggccagtga attcgagctc ggtaccctac agttgaagtc ggaagtttac 6721 atacacttaa gttggagtca ttaaaactcg tttttcaact actccacaaa tttcttgtta 6781 acaaacaata gttttggcaa gtcagttagg acatctactt tgtgcatgac acaagtcatt 6841 tttccaacaa ttgtttacag acagattatt tcacttataa ttcactgtat cacaattcca 6901 gtgggtcaga agtttacata cactaagttg actgtgcctt taaacagctt ggaaaattcc 6961 agaaaatgat gtcatggctt tagaagcttc tgatagacta attgacatca tttgagtcaa 7021 ttggaggtgt acctgtggat gtatttcaag gaattctgtg gaatgtgtgt cagttagggt 7081 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat atcgataagc 7141 tttgcaaaga tggataaag //
