| 质粒名称: | BB3cK_pGAP_23_pPFK300_Cas9 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 104910 |
| 存储实验室: | Brigitte Gasser |
|
载体骨架基本信息 |
|
| 载体名称: | Episomal ARS/CEN Backbone |
| 载体出品公司: | -- |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 酵母表达,CRISPR |
| 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 |
|
| 插入基因名称: | HH - FS23 linker - HDV |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | BbsI (未破坏) |
| 3' 克隆位点: | BbsI (未破坏) |
| 5' 测序引物 | pGAP fw |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | BB3cK_pGAP_23_pPFK300_Cas9 was a gift from Brigitte Gasser (Addgene plasmid # 104910 ; http://n2t.net/addgene:104910 ; RRID:Addgene_104910) |
| 参考文献部分: | CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris. Gassler T, Heistinger L, Mattanovich D, Gasser B, Prielhofer R. Methods Mol Biol. 2019;1923:211-225. doi: 10.1007/978-1-4939-9024-5_9. 10.1007/978-1-4939-9024-5_9 PubMed 30737742 |
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 BB3cK_pGAP_23_pPFK300_Cas9 8327 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION hCas9 under control of pPFK300 for direct cloning of HH-sgRNA-HDV
PCR products for episomal expression in P. pastoris and selection on
G418.
ACCESSION .
VERSION .
KEYWORDS BB3cK_pGAP_23_pPFK300_Cas9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8327)
AUTHORS Gassler T, Heistinger L, Mattanovich D, Gasser B, Prielhofer R.
TITLE CRISPR/Cas9-mediated homology directed genome editing in Pichia
pastoris
JOURNAL (2018) In Press
REFERENCE 2 (bases 1 to 8327)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8327
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin complement(70..658)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(150..169)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
promoter 745..1221
/label=GAP promoter
/note="promoter for Pichia pastoris
glyceraldehyde-3-phosphate dehydrogenase"
CDS 1988..6091
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 6104..6124
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
terminator 6196..6443
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
primer_bind complement(6212..6230)
/label=CYC1
/note="CYC1 transcription termination signal, reverse
primer"
gene 6463..7819
/label=kanMX
/note="yeast selectable marker conferring kanamycin
resistance (Wach et al., 1994)"
promoter 6463..6806
/label=TEF promoter
/note="Ashbya gossypii TEF promoter"
CDS 6807..7616
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind complement(6874..6893)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
terminator 7622..7819
/label=TEF terminator
/note="Ashbya gossypii TEF terminator"
misc_feature 7831..7
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
ORIGIN
1 agcgatcgcg ctgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
61 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
121 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
181 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
241 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
301 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
361 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
421 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
481 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
541 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
601 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
661 agatcctttg atcttttcta cggggtggag cgcaaaaaac cccgcccctg acagggcggg
721 gttttttcgc gatcggagga tccttttttg tagaaatgtc ttggtgtcct cgtccaatca
781 ggtagccatc tctgaaatat ctggctccgt tgcaactccg aacgacctgc tggcaacgta
841 aaattctccg gggtaaaact taaatgtgga gtaatggaac cagaaacgtc tcttcccttc
901 tctctccttc caccgcccgt taccgtccct aggaaatttt actctgctgg agagcttctt
961 ctacggcccc cttgcagcaa tgctcttccc agcattacgt tgcgggtaaa acggaggtcg
1021 tgtacccgac ctagcagccc agggatggaa aagtcccggc cgtcgctggc aataatagcg
1081 ggcggacgca tgtcatgaga ttattggaaa ccaccagaat cgaatataaa aggcgaacac
1141 ctttcccaat tttggtttct cctgacccaa agactttaaa tttaatttat ttgtccctat
1201 ttcaatcaat tgaacaacta tcaaaacacc atggcgtctt cggatccctc gaggaagacg
1261 cgcttttagt gtacatctga taatatagta ctaccacgta tgataatgta gagaataggc
1321 ttccttgtcg agtgtgtttg cagttttctt gagtttcaag gtttaaatgc tggtatatta
1381 gttcatcgaa ggtttcagcc aatagcacct taaatcaatc aaactaattc gactcttacg
1441 aaagagccta ctgtgtttag tatcgaagtc gtttaccttt catgttgaat agcttcctct
1501 ctgaccctaa catttcaaga tcctcctaaa gttacccgga ttgtgaaatt ctaatgatcc
1561 acctgcccaa tgcatttttt ctttattcag tttacctttt ttacctaata tacgagcttg
1621 ttaaagtaag tggcactgca atactaggct tattgttgat attatgatga atcgttttca
1681 caaacttgat ttcctgtgaa ctcaccatgt actaaggaaa aaaacatgca tcaccatctg
1741 aatatttgac cgctccgggg agcctgccat taggcaaaca ccaactagga acctacacca
1801 agttatgaag gagcttggaa aggattataa aaaatgcgat tacctaatac acacacgatg
1861 cctgagcggt taatgtgaaa tttcccggcc atcgatcgcc ccacaatcag actattttcg
1921 ctcatacaat aaaaagtctc ctgtttccac ccgagtatca ccagaacaaa gtaacaaata
1981 caaaaccatg gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggttg
2041 ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac
2101 cgatcgccac agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac
2161 ggccgaagcc acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg
2221 gatctgctac ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt
2281 ccataggctg gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat
2341 ctttggcaat atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct
2401 gaggaagaag cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct
2461 ggcgcatatg atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa
2521 cagcgatgtc gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga
2581 gaacccgatc aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa
2641 atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt
2701 tggtaatctt atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct
2761 ggccgaagat gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct
2821 gctggcccag atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga
2881 cgccattctg ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag
2941 cgctagtatg atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct
3001 tgtcagacag caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg
3061 ctacgccgga tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc
3121 catcttggaa aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct
3181 gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga
3241 actgcacgct atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga
3301 aaagattgag aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg
3361 aaattccaga ttcgcgtgga tgactcgcaa atcagaagag actatcactc cctggaactt
3421 cgaggaagtc gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt
3481 tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt
3541 cacagtttat aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc
3601 attcctgtct ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa
3661 agttaccgtg aaacagctca aagaggacta tttcaaaaag attgaatgtt tcgactctgt
3721 tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct
3781 gaaaatcatt aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga
3841 cattgtcctc acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac
3901 ttacgctcat ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg
3961 atgggggcgg ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac
4021 aatcctggat tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca
4081 tgatgactct ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga
4141 cagccttcac gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact
4201 gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa
4261 tatcgttatc gagatggccc gagagaacca aactacccag aagggacaga agaacagtag
4321 ggaaaggatg aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga
4381 acacccagtt gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa
4441 cggcagggac atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt
4501 ggatcatatc gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac
4561 aagatccgat aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa
4621 aatgaaaaat tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga
4681 taatctgact aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa
4741 aaggcagctt gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg
4801 catgaacacc aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct
4861 gaagtctaag ctcgtctcag atttcagaaa ggactttcag ttttataagg tgagagagat
4921 caacaattac caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat
4981 caaaaaatat cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt
5041 taggaaaatg atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt
5101 ttacagcaat attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg
5161 gaagcgacca cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag
5221 ggatttcgcg acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac
5281 cgaagtacag accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa
5341 gctgatcgca cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac
5401 agtcgcttac agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa
5461 aagcgtcaag gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc
5521 catcgacttt ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct
5581 tcccaagtac tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg
5641 cgagctgcag aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct
5701 ggccagccac tatgaaaagc tcaaagggtc ccccgaagat aatgagcaga agcagctgtt
5761 cgtggaacaa cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa
5821 aagagtgatc ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag
5881 ggataagccc atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt
5941 gggcgcgcct gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc
6001 tacaaaggag gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac
6061 aagaatcgac ctctctcagc tcggtggaga cagcagggct gaccccaaga agaagaggaa
6121 ggtgtgagct tcgtccgacg gcggcccgac gggtccgagg cctcggagat ccgtccccct
6181 tttcctttgt cgatatcatg taattagtta tgtcacgctt acattcacgc cctcccccca
6241 catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt
6301 tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc
6361 tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg
6421 gacgctcgaa ggctttaatt tgcaagctgg cgctaattca tggacatgga ggcccagaat
6481 accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg tcgcccgtac
6541 atttagccca tacatcccca tgtataatca tttgcatcca tacattttga tggccgcacg
6601 gcgcgaagca aaaattacgg ctcctcgctg cagacgtgcg agcagggaaa cgctcccctc
6661 acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa aggttaggat
6721 ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg atacagttct
6781 cacatcacat ccgaacataa acaaccatgg gtaaggaaaa gactcacgtt tcgaggccgc
6841 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
6901 ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc
6961 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
7021 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
7081 catggttact caccactgcg atccccggca aaacagcatt ccaggtatta gaagaatatc
7141 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
7201 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
7261 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
7321 ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg gattcagtcg
7381 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
7441 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
7501 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
7561 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag
7621 tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat
7681 tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat
7741 catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg
7801 aatgctggtc gctatactgg cttccttttc atcacgtgct ataaaaataa ttataattta
7861 aattttttaa tataaatata taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa
7921 aatagttttt gttttccgaa gatgtaaaag actctagggg gatcgccaac aaatactacc
7981 ttttatcttg ctcttcctgc tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt
8041 agaacaccac acacgaaaat cctgtgattt tacattttac ttatcgttaa tcgaatgtat
8101 atctatttaa tctgcttttc ttgtctaata aatatatatg taaagtacgc tttttgttga
8161 aattttttaa acctttgttt attttttttt cttcattccg taactcttct accttcttta
8221 tttactttct aaaatccaaa tacaaaacat aaaaataaat aaacacagag taaattccca
8281 aattattcca tcattaaaag atacgaggcg cgtgtaagtt acaggca
//
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