| 质粒名称: | BB3cH_pGAP_23*_pPFK300_Cas9 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 104912 |
| 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: | 潮霉素 |
| 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: | 潮霉素 |
| 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: | 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: | BB3cH_pGAP_23*_pPFK300_Cas9 was a gift from Brigitte Gasser (Addgene plasmid # 104912 ; http://n2t.net/addgene:104912 ; RRID:Addgene_104912) |
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 BB3cH_pGAP_23*_pPFK300_Cas9 8546 bp ds-DNA circular SYN 07-AUG-2018
DEFINITION hCas9 under control of pPFK300 for direct cloning of HH-sgRNA-HDV
PCR products for episomal expression in P. pastoris and selection on
Hyg.
ACCESSION .
VERSION .
KEYWORDS BB3cH_pGAP_23*_pPFK300_Cas9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8546)
TITLE CRISPR in Pichia pastoris
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 8546)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 7, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8546
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin complement(62..650)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(142..161)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
promoter 737..1213
/label=GAP promoter
/note="promoter for Pichia pastoris
glyceraldehyde-3-phosphate dehydrogenase"
CDS 1980..6083
/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 6096..6116
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
terminator 6188..6435
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
primer_bind complement(6204..6222)
/label=CYC1
/note="CYC1 transcription termination signal, reverse
primer"
gene 6455..8030
/label=hphMX6
/note="yeast selectable marker conferring hygromycin
resistance"
promoter 6455..6798
/label=TEF promoter
/note="Ashbya gossypii TEF promoter"
CDS 6799..7827
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDLGGR
GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
KE"
terminator 7833..8030
/label=TEF terminator
/note="Ashbya gossypii TEF terminator"
misc_feature 8042..8545
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
ORIGIN
1 cgctgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
481 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
661 tgatcttttc tacggggtgg agcgcaaaaa accccgcccc tgacagggcg gggttttttc
721 gcgatcggag gatccttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca
781 tctctgaaat atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc
841 cggggtaaaa cttaaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct
901 tccaccgccc gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc
961 cccttgcagc aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg
1021 acctagcagc ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg
1081 catgtcatga gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca
1141 attttggttt ctcctgaccc aaagacttta aatttaattt atttgtccct atttcaatca
1201 attgaacaac tatcaaaaca ccatggcgtc ttcggatccc tcgaggaaga cgcgctttta
1261 gtgtacatct gataatatag tactaccacg tatgataatg tagagaatag gcttccttgt
1321 cgagtgtgtt tgcagttttc ttgagtttca aggtttaaat gctggtatat tagttcatcg
1381 aaggtttcag ccaatagcac cttaaatcaa tcaaactaat tcgactctta cgaaagagcc
1441 tactgtgttt agtatcgaag tcgtttacct ttcatgttga atagcttcct ctctgaccct
1501 aacatttcaa gatcctccta aagttacccg gattgtgaaa ttctaatgat ccacctgccc
1561 aatgcatttt ttctttattc agtttacctt ttttacctaa tatacgagct tgttaaagta
1621 agtggcactg caatactagg cttattgttg atattatgat gaatcgtttt cacaaacttg
1681 atttcctgtg aactcaccat gtactaagga aaaaaacatg catcaccatc tgaatatttg
1741 accgctccgg ggagcctgcc attaggcaaa caccaactag gaacctacac caagttatga
1801 aggagcttgg aaaggattat aaaaaatgcg attacctaat acacacacga tgcctgagcg
1861 gttaatgtga aatttcccgg ccatcgatcg ccccacaatc agactatttt cgctcataca
1921 ataaaaagtc tcctgtttcc acccgagtat caccagaaca aagtaacaaa tacaaaacca
1981 tggacaagaa gtactccatt gggctcgata tcggcacaaa cagcgtcggt tgggccgtca
2041 ttacggacga gtacaaggtg ccgagcaaaa aattcaaagt tctgggcaat accgatcgcc
2101 acagcataaa gaagaacctc attggcgccc tcctgttcga ctccggggag acggccgaag
2161 ccacgcggct caaaagaaca gcacggcgca gatatacccg cagaaagaat cggatctgct
2221 acctgcagga gatctttagt aatgagatgg ctaaggtgga tgactctttc ttccataggc
2281 tggaggagtc ctttttggtg gaggaggata aaaagcacga gcgccaccca atctttggca
2341 atatcgtgga cgaggtggcg taccatgaaa agtacccaac catatatcat ctgaggaaga
2401 agcttgtaga cagtactgat aaggctgact tgcggttgat ctatctcgcg ctggcgcata
2461 tgatcaaatt tcggggacac ttcctcatcg agggggacct gaacccagac aacagcgatg
2521 tcgacaaact ctttatccaa ctggttcaga cttacaatca gcttttcgaa gagaacccga
2581 tcaacgcatc cggagttgac gccaaagcaa tcctgagcgc taggctgtcc aaatcccggc
2641 ggctcgaaaa cctcatcgca cagctccctg gggagaagaa gaacggcctg tttggtaatc
2701 ttatcgccct gtcactcggg ctgaccccca actttaaatc taacttcgac ctggccgaag
2761 atgccaagct tcaactgagc aaagacacct acgatgatga tctcgacaat ctgctggccc
2821 agatcggcga ccagtacgca gacctttttt tggcggcaaa gaacctgtca gacgccattc
2881 tgctgagtga tattctgcga gtgaacacgg agatcaccaa agctccgctg agcgctagta
2941 tgatcaagcg ctatgatgag caccaccaag acttgacttt gctgaaggcc cttgtcagac
3001 agcaactgcc tgagaagtac aaggaaattt tcttcgatca gtctaaaaat ggctacgccg
3061 gatacattga cggcggagca agccaggagg aattttacaa atttattaag cccatcttgg
3121 aaaaaatgga cggcaccgag gagctgctgg taaagcttaa cagagaagat ctgttgcgca
3181 aacagcgcac tttcgacaat ggaagcatcc cccaccagat tcacctgggc gaactgcacg
3241 ctatcctcag gcggcaagag gatttctacc cctttttgaa agataacagg gaaaagattg
3301 agaaaatcct cacatttcgg ataccctact atgtaggccc cctcgcccgg ggaaattcca
3361 gattcgcgtg gatgactcgc aaatcagaag agactatcac tccctggaac ttcgaggaag
3421 tcgtggataa gggggcctct gcccagtcct tcatcgaaag gatgactaac tttgataaaa
3481 atctgcctaa cgaaaaggtg cttcctaaac actctctgct gtacgagtac ttcacagttt
3541 ataacgagct caccaaggtc aaatacgtca cagaagggat gagaaagcca gcattcctgt
3601 ctggagagca gaagaaagct atcgtggacc tcctcttcaa gacgaaccgg aaagttaccg
3661 tgaaacagct caaagaggac tatttcaaaa agattgaatg tttcgactct gttgaaatca
3721 gcggagtgga ggatcgcttc aacgcatccc tgggaacgta tcacgatctc ctgaaaatca
3781 ttaaagacaa ggacttcctg gacaatgagg agaacgagga cattcttgag gacattgtcc
3841 tcacccttac gttgtttgaa gatagggaga tgattgaaga acgcttgaaa acttacgctc
3901 atctcttcga cgacaaagtc atgaaacagc tcaagaggcg ccgatataca ggatgggggc
3961 ggctgtcaag aaaactgatc aatgggatcc gagacaagca gagtggaaag acaatcctgg
4021 attttcttaa gtccgatgga tttgccaacc ggaacttcat gcagttgatc catgatgact
4081 ctctcacctt taaggaggac atccagaaag cacaagtttc tggccagggg gacagccttc
4141 acgagcacat cgctaatctt gcaggtagcc cagctatcaa aaagggaata ctgcagaccg
4201 ttaaggtcgt ggatgaactc gtcaaagtaa tgggaaggca taagcccgag aatatcgtta
4261 tcgagatggc ccgagagaac caaactaccc agaagggaca gaagaacagt agggaaagga
4321 tgaagaggat tgaagagggt ataaaagaac tggggtccca aatccttaag gaacacccag
4381 ttgaaaacac ccagcttcag aatgagaagc tctacctgta ctacctgcag aacggcaggg
4441 acatgtacgt ggatcaggaa ctggacatca atcggctctc cgactacgac gtggatcata
4501 tcgtgcccca gtcttttctc aaagatgatt ctattgataa taaagtgttg acaagatccg
4561 ataaaaatag agggaagagt gataacgtcc cctcagaaga agttgtcaag aaaatgaaaa
4621 attattggcg gcagctgctg aacgccaaac tgatcacaca acggaagttc gataatctga
4681 ctaaggctga acgaggtggc ctgtctgagt tggataaagc cggcttcatc aaaaggcagc
4741 ttgttgagac acgccagatc accaagcacg tggcccaaat tctcgattca cgcatgaaca
4801 ccaagtacga tgaaaatgac aaactgattc gagaggtgaa agttattact ctgaagtcta
4861 agctcgtctc agatttcaga aaggactttc agttttataa ggtgagagag atcaacaatt
4921 accaccatgc gcatgatgcc tacctgaatg cagtggtagg cactgcactt atcaaaaaat
4981 atcccaagct tgaatctgaa tttgtttacg gagactataa agtgtacgat gttaggaaaa
5041 tgatcgcaaa gtctgagcag gaaataggca aggccaccgc taagtacttc ttttacagca
5101 atattatgaa ttttttcaag accgagatta cactggccaa tggagagatt cggaagcgac
5161 cacttatcga aacaaacgga gaaacaggag aaatcgtgtg ggacaagggt agggatttcg
5221 cgacagtccg gaaggtcctg tccatgccgc aggtgaacat cgttaaaaag accgaagtac
5281 agaccggagg cttctccaag gaaagtatcc tcccgaaaag gaacagcgac aagctgatcg
5341 cacgcaaaaa agattgggac cccaagaaat acggcggatt cgattctcct acagtcgctt
5401 acagtgtact ggttgtggcc aaagtggaga aagggaagtc taaaaaactc aaaagcgtca
5461 aggaactgct gggcatcaca atcatggagc gatcaagctt cgaaaaaaac cccatcgact
5521 ttctcgaggc gaaaggatat aaagaggtca aaaaagacct catcattaag cttcccaagt
5581 actctctctt tgagcttgaa aacggccgga aacgaatgct cgctagtgcg ggcgagctgc
5641 agaaaggtaa cgagctggca ctgccctcta aatacgttaa tttcttgtat ctggccagcc
5701 actatgaaaa gctcaaaggg tcccccgaag ataatgagca gaagcagctg ttcgtggaac
5761 aacacaaaca ctaccttgat gagatcatcg agcaaataag cgaattctcc aaaagagtga
5821 tcctcgccga cgctaacctc gataaggtgc tttctgctta caataagcac agggataagc
5881 ccatcaggga gcaggcagaa aacattatcc acttgtttac tctgaccaac ttgggcgcgc
5941 ctgcagcctt caagtacttc gacaccacca tagacagaaa gcggtacacc tctacaaagg
6001 aggtcctgga cgccacactg attcatcagt caattacggg gctctatgaa acaagaatcg
6061 acctctctca gctcggtgga gacagcaggg ctgaccccaa gaagaagagg aaggtgtgag
6121 cttcgtccga cggcggcccg acgggtccga ggcctcggag atccgtcccc cttttccttt
6181 gtcgatatca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct
6241 ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata
6301 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga
6361 cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg
6421 aaggctttaa tttgcaagct ggcgctaatt catggacatg gaggcccaga ataccctcct
6481 tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc
6541 catacatccc catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag
6601 caaaaattac ggctcctcgc tgcagacgtg cgagcaggga aacgctcccc tcacagacgc
6661 gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact
6721 gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac
6781 atccgaacat aaacaaccat gggtaaaaag cctgaactca ccgcgacgtc tgtcgagaag
6841 tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa
6901 tctcgtgctt tcagcttcga tttaggaggg cgtggatatg tcctgcgggt aaatagctgc
6961 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg
7021 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc
7081 cgtgcacagg gtgtcacgtt gcaagacctc cctgaaaccg aactgcccgc tgttctgcag
7141 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
7201 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
7261 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
7321 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
7381 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
7441 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
7501 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
7561 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
7621 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc
7681 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
7741 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
7801 agcactcgtc cgagggcaaa ggaataatca gtactgacaa taaaaagatt cttgttttca
7861 agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta
7921 gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc
7981 agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg gcttcctttt
8041 catcacgtgc tataaaaata attataattt aaatttttta atataaatat ataaattaaa
8101 aatagaaagt aaaaaaagaa attaaagaaa aaatagtttt tgttttccga agatgtaaaa
8161 gactctaggg ggatcgccaa caaatactac cttttatctt gctcttcctg ctctcaggta
8221 ttaatgccga attgtttcat cttgtctgtg tagaacacca cacacgaaaa tcctgtgatt
8281 ttacatttta cttatcgtta atcgaatgta tatctattta atctgctttt cttgtctaat
8341 aaatatatat gtaaagtacg ctttttgttg aaatttttta aacctttgtt tatttttttt
8401 tcttcattcc gtaactcttc taccttcttt atttactttc taaaatccaa atacaaaaca
8461 taaaaataaa taaacacaga gtaaattccc aaattattcc atcattaaaa gatacgaggc
8521 gcgtgtaagt tacaggcaag cgatcg
//
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