质粒名称: | pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283) |
---|---|
出品公司: | addgene |
目录编号: | 115138 |
存储实验室: | Joseph Bondy-Denomy, Keith Joung, Benjamin Kleinstiver |
载体骨架基本信息 |
|
载体名称: | JDS246 (Plasmid #43861) |
载体出品公司: | Joung lab (Addgene Plasmid #43861) |
完整质粒大小: | 4416bp |
载体类型: | 哺乳细胞表达 |
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 |
|
插入基因名称: | human codon optimized AcrVA2 anti-CRISPR protein / AAS2283 |
物种来源: | Synthetic |
标签/融合蛋白: | NLS(SV40) (C terminal on insert) |
克隆方法: | Gibson Cloning |
5’测序引物 | CMV-F |
3’测序引物 | BGH-rev |
文献引用方法 |
|
材料和方法部分: | pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283) was a gift from Joseph Bondy-Denomy & Keith Joung & Benjamin Kleinstiver (Addgene plasmid # 115138 ; http://n2t.net/addgene:115138 ; RRID:Addgene_115138) |
参考文献部分: | Discovery of widespread Type I and Type V CRISPR-Cas inhibitors. Marino ND, Zhang JY, Borges AL, Sousa AA, Leon LM, Rauch BJ, Walton RT, Berry JD, Joung JK, Kleinstiver BP, Bondy-Denomy J. Science. 2018 Sep 6. pii: science.aau5174. doi: 10.1126/science.aau5174. 10.1126/science.aau5174 PubMed 30190308 |
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 pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283) 4416 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CMV-T7 promoter expression plasmid for human codon optimized AcrVA2 anti-CRISPR protein with C-terminal NLS . ACCESSION . VERSION . KEYWORDS pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4416) AUTHORS Marino ND, Zhang JY, Borges AL, Sousa AA, Leon LM, Rauch BJ, Walton RT, Berry JD, Joung JK, Kleinstiver BP, Bondy-Denomy J TITLE Discovery of widespread Type I and Type V CRISPR-Cas inhibitors. JOURNAL Science. 2018 Sep 6. pii: science.aau5174. doi: 10.1126/science.aau5174. PUBMED 30190308 REFERENCE 2 (bases 1 to 4416) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..4416 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 994..1014 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1023..1040 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1063..1080) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1069..1293 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(1364..1380) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1364..1380) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1377..1399) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 1388..1404 /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(1412..1442) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1457..1478 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(1595..1612) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1766..2354) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1846..1865) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2525..3385) /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 3148..3167 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3386..3490) /gene="bla" /label=AmpR promoter primer_bind complement(3569..3588) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 3760..4139 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 4140..4343 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 4294..4314 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 4385..4404 /label=T7 /note="T7 promoter, forward primer" promoter 4385..4403 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 4411..4 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" ORIGIN 1 atgggccatc acaccatcgc ccgcatgaat gctttcaaca aggcttttgc aaacgctaaa 61 gactgctaca aaaaaatgca agcctggcac ctgttgaaca aaccgaaaca cgcattcttt 121 cctatgcaga acacgccagc cctggataac ggcctcgctg cgctttatga actccggggg 181 ggaaaggaag atgcacatat tttgtctatt ctcagcagat tgtacttgta cggagcgtgg 241 cggaatacat tgggcatata ccagttggac gaggagatca ttaaggactg caaggagctt 301 cccgacgata caccgacgag catattcctc aacttgcctg actggtgtgt atatgtagat 361 atatccagtg ctcagatcgc cactttcgac gacggcgtag cgaagcacat caaaggtttc 421 tgggccatat acgatattgt tgaaatgaat ggaatcaacc atgacgtatt ggacttcgtt 481 gtagatactg acaccgacga taatgtgtac gttccgcagc ctttcatttt gtcatctggt 541 caatccgtgg cagaagtgct tgactatgga gcaagtttgt tcgacgacga tactagcaat 601 acgcttatta agggcctcct gccgtacttg ctctggcttt gcgtagcaga acccgacatc 661 acttacaaag gtttgccagt ctcaagagag gaacttactc gaccgaaaca ttcaatcaat 721 aaaaaaacgg gcgcgtttgt gacacctagt gagccattca tctaccagat aggagaacgg 781 ctgggaagtg aggttaggag gtatcagagc attatagatg gggagcaaaa gcgcaatcga 841 ccacatacaa agcgccctca tattcgccgc ggtcactggc atggatattg gcaagggact 901 ggacaggcga aggagtttcg agttcgatgg caacccgctg tgttcgtgaa tagcggaaga 961 gtatcatccg gatccggcgg tggaggcagt gggcccaaga agaagaggaa agtctgaccg 1021 gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact gtgccttcta 1081 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 1141 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 1201 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 1261 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg 1321 gctcgatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 1381 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 1441 aagcctaggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 1501 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 1561 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 1621 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 1681 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 1741 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 1801 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 1861 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 1921 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 1981 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 2041 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 2101 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 2161 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 2221 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 2281 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 2341 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 2401 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 2461 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 2521 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 2581 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 2641 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 2701 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 2761 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 2821 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 2881 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 2941 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 3001 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 3061 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 3121 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 3181 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 3241 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 3301 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 3361 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 3421 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 3481 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatcgat 3541 ctcccgatcc cctagggtcg actctcagta caatctgctc tgatgccgca tagttaagcc 3601 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag 3661 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt 3721 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt 3781 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta 3841 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt 3901 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg 3961 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta 4021 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga 4081 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg 4141 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 4201 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 4261 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 4321 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag agatccgcgg 4381 ccgctaatac gactcactat agggagagcc gccacc //