质粒名称: | pcNDA-NES-PhoCl2c-mCherry |
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出品公司: | 爱迪基因 |
目录编号: | 164037 |
存储实验室: | Robert Campbell |
载体骨架基本信息 |
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载体名称: | pcDNA3.1+ |
载体抗性: | 氨苄青霉素 |
空载体大小: | 5350 bp |
完整质粒大小: | 6892 bp |
载体类型: | 哺乳细胞表达 |
筛选标记: | 新霉素(G418) |
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: | NES-PhoCl2c-mCherry |
插入大小: | 1542 bp |
启动子 | CMV promotor |
标签/融合蛋白: | kozak sequence(插入件上的N端子) |
克隆方法: | Gibson克隆 |
5' 测序引物 | CG CAAATGGGCG GTAGGCGTG |
3' 测序引物 | CCTCGACTG TGCCTTCTA |
文献引用方法 |
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材料和方法部分: | pcNDA-NES-PhoCl2c-mCherry was a gift from Robert Campbell (Addgene plasmid # 164037 ; http://n2t.net/addgene:164037 ; RRID:Addgene_164037) |
参考文献部分: | Photocleavable proteins that undergo fast and efficient dissociation. Lu X, Wen Y, Zhang S, Zhang W, Chen Y, Shen Y, Lemieux MJ, Campbell RE. Chem Sci. 2021 May 31;12(28):9658-9672. doi: 10.1039/d1sc01059j. eCollection 2021 Jul 21. 10.1039/d1sc01059j PubMed 34349937 |
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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 pcNDA-NES-PhoCl2c-mCherry 6895 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Optogenetic control of protein localization by PhoCl2c. ACCESSION . VERSION . KEYWORDS pcNDA-NES-PhoCl2c-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6895) AUTHORS Lu X, Wen Y, Zhang S, Zhang W, Chen Y, Shen Y, Lemieux MJ, Campbell RE TITLE Improved Photocleavable Proteins with Faster and More Efficient Dissociation JOURNAL bioRxiv, December 10, 2020. REFERENCE 2 (bases 1 to 6895) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6895 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1741..2448 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(1883..1901) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2151..2170 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(2486..2503) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2492..2716 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2762..3190 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2849..2868) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3059..3080 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3199..3219) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3204..3533 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3384..3519 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3446..3465 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3600..4394 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3654..3673) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4264..4283 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4568..4689 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4605..4624) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4659..4678 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4738..4754) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4738..4754) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4751..4773) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4762..4778 /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(4786..4816) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4831..4852 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4969..4986) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5140..5728) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5220..5239) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5899..6759) /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 6522..6541 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6760..6864) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 901 gccaccatga acctggtgga cctgcagaag aagctggagg agctggagct ggacgagcag 961 cagggaggca agcttagcgt gatccctgac tacttcaagc agagcttccc cgagggctac 1021 agctgggagc gcagcatgac ctacgaggac ggcggcatct gcatcgccac caacgacatc 1081 acaatggagg gggacagctt catcaacaag atccacttcc agggcacgaa cttccccccc 1141 aacggccccg tgatgcagaa gaggaccgtg ggctgggagg ccagcaccga gaagatgtac 1201 gagcgcgacg gcgtgctgaa gggcgacgtg aagatgaagc tgctgctgaa gggcggcggc 1261 cactatcgcg gcgactaccg caccacctac aaggtcaagc agaagcccgt aaagctgccc 1321 gactgccact tcgtggacca ccgcatcgag atcctgagcc acgacaagga ctacaacaag 1381 gtgaagctgt acgagcacgc cgtggccaag acttccaccg acagcatgga cgagctgtac 1441 aagggtggca gcggtggcat ggtgagcaag ggcgaggaga ccattacaag cgtgatcaag 1501 cctgacatga agaacaagct gcgcatggag ggcaacgtga acggccacgc cttcgtgatc 1561 gagggcgagg gcagcggcaa gcccttcgag ggcatccaga cgattgattt ggaggtgaag 1621 gagggcgccc cgctgccctt cgcctacgac atcctgacca ccgccttcca ctacggcaac 1681 cgcgtgttca ccaagtaccc acggggtacc ggaggcgggg gcagcggagg cgggggcagc 1741 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg 1801 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc 1861 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc 1921 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc 1981 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg 2041 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc 2101 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg 2161 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc 2221 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag 2281 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac 2341 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc 2401 gccgagggcc gccactccac cggtggcatg gacgagctgt acaagtaact cgagtctaga 2461 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt 2521 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc 2581 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt 2641 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat 2701 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc 2761 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2821 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2881 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2941 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 3001 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 3061 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 3121 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 3181 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc 3241 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt 3301 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 3361 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 3421 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg 3481 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc 3541 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca 3601 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg 3661 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag 3721 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc 3781 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc 3841 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg 3901 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc 3961 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca 4021 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag 4081 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg 4141 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg 4201 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca 4261 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc 4321 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg 4381 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct 4441 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt 4501 tttccgggac accggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc 4561 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 4621 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 4681 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc 4741 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 4801 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 4861 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 4921 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4981 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 5041 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 5101 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 5161 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 5221 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 5281 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 5341 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 5401 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 5461 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 5521 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 5581 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 5641 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 5701 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 5761 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 5821 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 5881 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 5941 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 6001 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 6061 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 6121 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 6181 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 6241 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 6301 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 6361 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 6421 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 6481 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 6541 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 6601 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 6661 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 6721 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 6781 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 6841 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc //
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