| 质粒名称: | BB3cK_pGAP_23*_pTEF_Cas9 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 104909 |
| 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*_pTEF_Cas9 was a gift from Brigitte Gasser (Addgene plasmid # 104909 ; http://n2t.net/addgene:104909 ; RRID:Addgene_104909) |
| 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*_pTEF_Cas9 8516 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION hCas9 under control of Tef1 for direct cloning of HH-sgRNA-HDV PCR
products and episomal expression in P. pastoris and G418 selection.
ACCESSION .
VERSION .
KEYWORDS BB3cK_pGAP_23*_pTEF_Cas9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8516)
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 8516)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8516
/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"
promoter 1763..2169
/gene="S. cerevisiae TEF1"
/label=TEF1 promoter
/note="promoter for EF-1-alpha"
CDS 2177..6280
/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 6293..6313
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
terminator 6385..6632
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
primer_bind complement(6401..6419)
/label=CYC1
/note="CYC1 transcription termination signal, reverse
primer"
gene 6652..8008
/label=kanMX
/note="yeast selectable marker conferring kanamycin
resistance (Wach et al., 1994)"
promoter 6652..6995
/label=TEF promoter
/note="Ashbya gossypii TEF promoter"
CDS 6996..7805
/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(7063..7082)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
terminator 7811..8008
/label=TEF terminator
/note="Ashbya gossypii TEF terminator"
misc_feature 8020..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 agcacacacc atagcttcaa aatgtttcta ctcctttttt
1801 actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac
1861 agcatactaa atttcccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg
1921 tttggaaaag aaaaaagcga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa
1981 atttttatca cgtttctttt tcttgaaaat tttttttttt gatttttttc tctttcgatg
2041 acctcccatt gatatttaag ttaataaacg gtgttcaatt tctcaagttt cagtttcatt
2101 tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc
2161 taatctaagg gcggtcatgg acaagaagta ctccattggg ctcgatatcg gcacaaacag
2221 cgtcggttgg gccgtcatta cggacgagta caaggtgccg agcaaaaaat tcaaagttct
2281 gggcaatacc gatcgccaca gcataaagaa gaacctcatt ggcgccctcc tgttcgactc
2341 cggggagacg gccgaagcca cgcggctcaa aagaacagca cggcgcagat atacccgcag
2401 aaagaatcgg atctgctacc tgcaggagat ctttagtaat gagatggcta aggtggatga
2461 ctctttcttc cataggctgg aggagtcctt tttggtggag gaggataaaa agcacgagcg
2521 ccacccaatc tttggcaata tcgtggacga ggtggcgtac catgaaaagt acccaaccat
2581 atatcatctg aggaagaagc ttgtagacag tactgataag gctgacttgc ggttgatcta
2641 tctcgcgctg gcgcatatga tcaaatttcg gggacacttc ctcatcgagg gggacctgaa
2701 cccagacaac agcgatgtcg acaaactctt tatccaactg gttcagactt acaatcagct
2761 tttcgaagag aacccgatca acgcatccgg agttgacgcc aaagcaatcc tgagcgctag
2821 gctgtccaaa tcccggcggc tcgaaaacct catcgcacag ctccctgggg agaagaagaa
2881 cggcctgttt ggtaatctta tcgccctgtc actcgggctg acccccaact ttaaatctaa
2941 cttcgacctg gccgaagatg ccaagcttca actgagcaaa gacacctacg atgatgatct
3001 cgacaatctg ctggcccaga tcggcgacca gtacgcagac ctttttttgg cggcaaagaa
3061 cctgtcagac gccattctgc tgagtgatat tctgcgagtg aacacggaga tcaccaaagc
3121 tccgctgagc gctagtatga tcaagcgcta tgatgagcac caccaagact tgactttgct
3181 gaaggccctt gtcagacagc aactgcctga gaagtacaag gaaattttct tcgatcagtc
3241 taaaaatggc tacgccggat acattgacgg cggagcaagc caggaggaat tttacaaatt
3301 tattaagccc atcttggaaa aaatggacgg caccgaggag ctgctggtaa agcttaacag
3361 agaagatctg ttgcgcaaac agcgcacttt cgacaatgga agcatccccc accagattca
3421 cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat ttctacccct ttttgaaaga
3481 taacagggaa aagattgaga aaatcctcac atttcggata ccctactatg taggccccct
3541 cgcccgggga aattccagat tcgcgtggat gactcgcaaa tcagaagaga ctatcactcc
3601 ctggaacttc gaggaagtcg tggataaggg ggcctctgcc cagtccttca tcgaaaggat
3661 gactaacttt gataaaaatc tgcctaacga aaaggtgctt cctaaacact ctctgctgta
3721 cgagtacttc acagtttata acgagctcac caaggtcaaa tacgtcacag aagggatgag
3781 aaagccagca ttcctgtctg gagagcagaa gaaagctatc gtggacctcc tcttcaagac
3841 gaaccggaaa gttaccgtga aacagctcaa agaggactat ttcaaaaaga ttgaatgttt
3901 cgactctgtt gaaatcagcg gagtggagga tcgcttcaac gcatccctgg gaacgtatca
3961 cgatctcctg aaaatcatta aagacaagga cttcctggac aatgaggaga acgaggacat
4021 tcttgaggac attgtcctca cccttacgtt gtttgaagat agggagatga ttgaagaacg
4081 cttgaaaact tacgctcatc tcttcgacga caaagtcatg aaacagctca agaggcgccg
4141 atatacagga tgggggcggc tgtcaagaaa actgatcaat gggatccgag acaagcagag
4201 tggaaagaca atcctggatt ttcttaagtc cgatggattt gccaaccgga acttcatgca
4261 gttgatccat gatgactctc tcacctttaa ggaggacatc cagaaagcac aagtttctgg
4321 ccagggggac agccttcacg agcacatcgc taatcttgca ggtagcccag ctatcaaaaa
4381 gggaatactg cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa
4441 gcccgagaat atcgttatcg agatggcccg agagaaccaa actacccaga agggacagaa
4501 gaacagtagg gaaaggatga agaggattga agagggtata aaagaactgg ggtcccaaat
4561 ccttaaggaa cacccagttg aaaacaccca gcttcagaat gagaagctct acctgtacta
4621 cctgcagaac ggcagggaca tgtacgtgga tcaggaactg gacatcaatc ggctctccga
4681 ctacgacgtg gatcatatcg tgccccagtc ttttctcaaa gatgattcta ttgataataa
4741 agtgttgaca agatccgata aaaatagagg gaagagtgat aacgtcccct cagaagaagt
4801 tgtcaagaaa atgaaaaatt attggcggca gctgctgaac gccaaactga tcacacaacg
4861 gaagttcgat aatctgacta aggctgaacg aggtggcctg tctgagttgg ataaagccgg
4921 cttcatcaaa aggcagcttg ttgagacacg ccagatcacc aagcacgtgg cccaaattct
4981 cgattcacgc atgaacacca agtacgatga aaatgacaaa ctgattcgag aggtgaaagt
5041 tattactctg aagtctaagc tcgtctcaga tttcagaaag gactttcagt tttataaggt
5101 gagagagatc aacaattacc accatgcgca tgatgcctac ctgaatgcag tggtaggcac
5161 tgcacttatc aaaaaatatc ccaagcttga atctgaattt gtttacggag actataaagt
5221 gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa
5281 gtacttcttt tacagcaata ttatgaattt tttcaagacc gagattacac tggccaatgg
5341 agagattcgg aagcgaccac ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga
5401 caagggtagg gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt
5461 taaaaagacc gaagtacaga ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa
5521 cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg gcggattcga
5581 ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa
5641 aaaactcaaa agcgtcaagg aactgctggg catcacaatc atggagcgat caagcttcga
5701 aaaaaacccc atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat
5761 cattaagctt cccaagtact ctctctttga gcttgaaaac ggccggaaac gaatgctcgc
5821 tagtgcgggc gagctgcaga aaggtaacga gctggcactg ccctctaaat acgttaattt
5881 cttgtatctg gccagccact atgaaaagct caaagggtcc cccgaagata atgagcagaa
5941 gcagctgttc gtggaacaac acaaacacta ccttgatgag atcatcgagc aaataagcga
6001 attctccaaa agagtgatcc tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa
6061 taagcacagg gataagccca tcagggagca ggcagaaaac attatccact tgtttactct
6121 gaccaacttg ggcgcgcctg cagccttcaa gtacttcgac accaccatag acagaaagcg
6181 gtacacctct acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct
6241 ctatgaaaca agaatcgacc tctctcagct cggtggagac agcagggctg accccaagaa
6301 gaagaggaag gtgtgagctt cgtccgacgg cggcccgacg ggtccgaggc ctcggagatc
6361 cgtccccctt ttcctttgtc gatatcatgt aattagttat gtcacgctta cattcacgcc
6421 ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc
6481 ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc
6541 ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga
6601 aggttttggg acgctcgaag gctttaattt gcaagctggc gctaattcat ggacatggag
6661 gcccagaata ccctccttga cagtcttgac gtgcgcagct caggggcatg atgtgactgt
6721 cgcccgtaca tttagcccat acatccccat gtataatcat ttgcatccat acattttgat
6781 ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc agacgtgcga gcagggaaac
6841 gctcccctca cagacgcgtt gaattgtccc cacgccgcgc ccctgtagag aaatataaaa
6901 ggttaggatt tgccactgag gttcttcttt catatacttc cttttaaaat cttgctagga
6961 tacagttctc acatcacatc cgaacataaa caaccatggg taaggaaaag actcacgttt
7021 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
7081 ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag
7141 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
7201 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
7261 ctgatgatgc atggttactc accactgcga tccccggcaa aacagcattc caggtattag
7321 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
7381 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
7441 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
7501 atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg
7561 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
7621 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
7681 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
7741 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
7801 tctaatcagt actgacaata aaaagattct tgttttcaag aacttgtcat ttgtatagtt
7861 tttttatatt gtagttgttc tattttaatc aaatgttagc gtgatttata ttttttttcg
7921 cctcgacatc atctgcccag atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa
7981 tcgtatgtga atgctggtcg ctatactggc ttccttttca tcacgtgcta taaaaataat
8041 tataatttaa attttttaat ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat
8101 taaagaaaaa atagtttttg ttttccgaag atgtaaaaga ctctaggggg atcgccaaca
8161 aatactacct tttatcttgc tcttcctgct ctcaggtatt aatgccgaat tgtttcatct
8221 tgtctgtgta gaacaccaca cacgaaaatc ctgtgatttt acattttact tatcgttaat
8281 cgaatgtata tctatttaat ctgcttttct tgtctaataa atatatatgt aaagtacgct
8341 ttttgttgaa attttttaaa cctttgttta tttttttttc ttcattccgt aactcttcta
8401 ccttctttat ttactttcta aaatccaaat acaaaacata aaaataaata aacacagagt
8461 aaattcccaa attattccat cattaaaaga tacgaggcgc gtgtaagtta caggca
//
