• 规格：20μl质粒
• 启动子：TEF1
• 原核抗性：氨苄青霉素Amp
• 筛选标记：TRP1
• 克隆菌株：大肠杆菌DH5α
• 质粒简介：               p414-TEF1p-Cas9-CYC1t是一个酵母酵母基因编辑Cas9表达质粒。
• 质粒图谱1： png/1-1P10614561M18.png

质粒序列： 点击查看序列

LOCUS       Exported                9524 bp ds-DNA     circular SYN 06-JAN-2018

DEFINITION  synthetic circular DNA

FEATURES             Location/Qualifiers

     source          1..9524

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     misc_feature    69..572

                     /label=CEN/ARS

                     /note="S. cerevisiae CEN6 centromere fused to an 

                     autonomously replicating sequence"

     promoter        609..713

                     /gene="bla"

                     /label=AmpR promoter

     CDS             714..1574

                     /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"

     rep_origin      1745..2333

                     /direction=RIGHT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 

                     replication"

     promoter        2792..3190

                     /gene="S. cerevisiae TEF1"

                     /label=TEF1 promoter

                     /note="promoter for EF-1-alpha"

     CDS             3222..7325

                     /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             7338..7358

                     /codon_start=1

                     /product="nuclear localization signal of SV40 (simian virus

                     40) large T antigen"

                     /label=SV40 NLS

                     /translation="PKKKRKV"

     terminator      7374..7621

                     /gene="S. cerevisiae CYC1"

                     /label=CYC1 terminator

                     /note="transcription terminator for CYC1"

     rep_origin      7829..8284

                     /direction=RIGHT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow 

                     indicates direction of (+) strand synthesis"

     CDS             complement(8383..9057)

                     /codon_start=1

                     /gene="S. cerevisiae TRP1"

                     /product="phosphoribosylanthranilate isomerase, required 

                     for tryptophan biosynthesis"

                     /label=TRP1

                     /note="yeast auxotrophic marker"

                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK

                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES

                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW

                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA

                     KK"

     promoter        complement(9058..9338)

                     /gene="S. cerevisiae TRP1"

                     /label=TRP1 promoter

ORIGIN

        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt

       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa

      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta

      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa

      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag

      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt

      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa

      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta

      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa

      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg

      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc

      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta

      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg

      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg

      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac

      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg

      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt

     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg

     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac

     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt

     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag

     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc

     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc

     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta

     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg

     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga

     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac

     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa

     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat

     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc

     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg

     1861 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc

     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg

     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg

     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa

     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg

     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga

     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct

     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca

     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc

     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg

     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc

     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca

     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tacctcactc

     2641 attaggcacc ccaggcttta cactttatgc ttccggctcc tatgttgtgt ggaattgtga

     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta

     2761 accctcacta aagggaacaa aagctggagc tcatagcttc aaaatgtttc tactcctttt

     2821 ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc

     2881 acagcatact aaatttcccc tctttcttcc tctagggtgt cgttaattac ccgtactaaa

     2941 ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa

     3001 aaatttttat cacgtttctt tttcttgaaa attttttttt tgattttttt ctctttcgat

     3061 gacctcccat tgatatttaa gttaataaac ggtcttcaat ttctcaagtt tcagtttcat

     3121 ttttcttgtt ctattacaac tttttttact tcttgctcat tagaaagaaa gcatagcaat

     3181 ctaatctaag ttttctagaa ctagtggatc ccccgggaaa aat