| 质粒名称: | pMaCTag-P05 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 120016 |
| 存储实验室: | Michael Knop |
|
载体骨架基本信息 |
|
| 载体名称: | pFA6a |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 2412 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | PCR模板 |
| 筛选标记: | 嘌呤霉素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | eGFP |
| 物种来源: | H. sapiens (human) |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | eGFP（主干上的C端） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | BamHI(未破坏) |
| 3' 克隆位点: | SpeI (未破坏) |
| 5' 测序引物 | SP6 |
| 3' 测序引物 | AAACCACAACTAGAATGCAGTG |
|
文献引用方法 |
|
| 材料和方法部分: | pMaCTag-P05 was a gift from Michael Knop (Addgene plasmid # 120016 ; http://n2t.net/addgene:120016 ; RRID:Addgene_120016) |
| 参考文献部分: | CRISPR-Cas12a-assisted PCR tagging of mammalian genes. Fueller J, Herbst K, Meurer M, Gubicza K, Kurtulmus B, Knopf JD, Kirrmaier D, Buchmuller BC, Pereira G, Lemberg MK, Knop M. J Cell Biol. 2020 Jun 1;219(6). pii: 151766. doi: 10.1083/jcb.201910210. 10.1083/jcb.201910210 PubMed 32406907 |
买家导航
pMaCTagP05载体基本信息
pMaCTagP05载体图谱质粒图谱和多克隆位点信息
pMaCTagP05载体简介
pMaCTagP05载体序列
LOCUS pMaCTag-P05 4807 bp ds-DNA circular SYN 08-JAN-2019
DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin
selection) with eGFP.
ACCESSION .
VERSION .
KEYWORDS pMaCTag-P05
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4807)
AUTHORS Fueller J, Meurer M, Herbst K, Gubicza K, Kurtulmus B, Knopf JD,
Kirrmaier D, Buchmuller B, Pereira G, Lemberg MK, Knop M
TITLE CRISPR/Cas12a-assisted PCR tagging of mammalian genes
JOURNAL bioRxiv
REFERENCE 2 (bases 1 to 4807)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jan 8, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..4807
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..19
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 1..18
/label=SP6
/note="SP6 promoter, forward primer"
CDS 81..797
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(123..144)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(384..403)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 731..752
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 804..925
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(841..860)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 895..914
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
protein_bind 932..965
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
promoter 972..1288
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 1139..1274
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 1201..1220
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
polyA_signal 1910..2134
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 2141..2381
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 2141..2161
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 2312..2331
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
protein_bind 2391..2424
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind complement(2461..2480)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(2462..2480)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2567..2584)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(2738..3326)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2818..2837)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(3497..4357)
/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 4120..4139
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(4358..4462)
/gene="bla"
/label=AmpR promoter
primer_bind 4530..4548
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(4586..4608)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 4708..4727
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
121 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
181 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
241 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
301 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
361 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
421 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
481 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
541 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
601 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
661 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
721 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
781 acgagctgta caagtaaact agtaacttgt ttattgcagc ttataatggt tacaaataaa
841 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
901 tgtccaaact catcaatgta tcttagtcga tataacttcg tatagcatac attatacgaa
961 gttatgtcga cggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
1021 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
1081 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
1141 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
1201 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
1261 cttttttgga ggcctaggct tttgcaaaga attcgccacc atgactgagt acaagcctac
1321 tgtgcgttta gccaccaggg atgatgtgcc acgagccgtg aggacactag cagccgcctt
1381 tgctgactac ccagctacaa ggcacaccgt ggaccctgac agacacattg aaagagtcac
1441 agaacttcag gagctgtttt taactagggt gggcctggat attggcaaag tctgggtggc
1501 cgatgatgga gcagcagttg cagtgtggac caccccagag tctgttgaag ctggagctgt
1561 ctttgcagaa attggaccaa gaatggccga gttatctggc agcaggctgg ctgctcaaca
1621 gcagatggaa ggattattag ctccccacag acccaaggag cctgcctggt tcctggcaac
1681 agttggagtt tctccagacc atcagggcaa gggcttagga tctgctgtgg tgctgcctgg
1741 tgtggaagca gctgagagag ctggggtgcc agcatttctg gagacatcag ctcctagaaa
1801 cctgcccttc tatgaacggc tgggcttcac agtgactgct gatgtggagg tgcctgaggg
1861 ccccagaacc tggtgcatga caagaaaacc aggagcctga ctgcagcgac tgtgccttct
1921 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
1981 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
2041 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
2101 agcaggcatg ctggggatgc ggtgggctct atggctcgag gagggcctat ttcccatgat
2161 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
2221 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
2281 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
2341 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg ataacttcgt
2401 atagcataca ttatacgaag ttatggtacc gatgcagcta gcccgcggat ctgccggtct
2461 ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc
2521 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
2581 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
2641 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
2701 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
2761 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
2821 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
2881 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
2941 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
3001 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
3061 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
3121 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
3181 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
3241 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
3301 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
3361 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
3421 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
3481 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
3541 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
3601 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
3661 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
3721 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
3781 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
3841 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
3901 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
3961 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
4021 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
4081 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
4141 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
4201 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
4261 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
4321 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
4381 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
4441 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
4501 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
4561 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
4621 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
4681 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
4741 atatggacat attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac
4801 acatacg
//
