基本信息
| 原核抗性: | 氨苄青霉素Amp |
| 克隆菌株: | 大肠杆菌DH5α |
| 培养条件: | 37℃ |
质粒简介
pwtCas9-bacteria是一个大肠杆菌Cas9四环素诱导表达质粒。质粒图谱
质粒序列
LOCUS Exported 6932 bp ds-DNA circular SYN 06-DEC-2017
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..6932
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(7..630)
/codon_start=1
/gene="tetR from transposon Tn10"
/product="tetracycline repressor TetR"
/label=TetR
/note="TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved by
adding tetracycline or doxycycline."
/translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
promoter 649..704
/gene="tetR"
/label=tetR/tetA promoters
/note="overlapping promoters for bacterial tetR and tetA"
protein_bind 685..703
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
RBS 722..733
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
CDS 740..4846
/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"
terminator 4870..4941
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 4957..4984
/label=T7Te terminator
/note="phage T7 early transcription terminator"
rep_origin complement(5151..5739)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
terminator 5827..5921
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
CDS complement(5951..6811)
/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"
promoter complement(6812..6916)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gcatgcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc
61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
601 aatcacttta cttttatcta atctagacat cattaattcc taatttttgt tgacactcta
661 tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaagatc
721 taaagaggag aaaggatcta tggataagaa atactcaata ggcttagata tcggcacaaa
781 tagcgtcgga tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt
841 tctgggaaat acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga
901 cagtggagag acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg
961 tcggaagaat cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga
1021 tgatagtttc tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga
1081 acgtcatcct atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac
1141 tatctatcat ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat
1201 ctatttggcc ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt
1261 aaatcctgat aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca
1321 attatttgaa gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc
1381 acgattgagt aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa
1441 aaatggctta tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc
1501 aaattttgat ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga
1561 tttagataat ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa
1621 gaatttatca gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa
1681 ggctccccta tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct
1741 tttaaaagct ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca
1801 atcaaaaaac ggatatgcag gttatattga tgggggagct agccaagaag aattttataa
1861 atttatcaaa ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa
1921 tcgtgaagat ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat
1981 tcacttgggt gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa
2041 agacaatcgt gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc
2101 attggcgcgt ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac
2161 cccatggaat tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg
2221 catgacaaac tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct
2281 ttatgagtat tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat
2341 gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa
2401 aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg
2461 ttttgatagt gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta
2521 ccatgatttg ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga
2581 tatcttagag gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga
2641 aagacttaaa acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg
2701 ccgttatact ggttggggac gtttgtctcg aaaattgatt aatggtatta gggataagca
2761 atctggcaaa acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat
2821 gcagctgatc catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc
2881 tggacaaggc gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa
2941 aaaaggtatt ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca
3001 taagccagaa aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca
3061 gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca
3121 gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta
3181 ttatctccaa aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag
3241 tgattatgat gtcgatcaca ttgttccaca aagtttcctt aaagacgatt caatagacaa
3301 taaggtctta acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga
3361 agtagtcaaa aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca
3421 acgtaagttt gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc
3481 tggttttatc aaacgccaat tggttgaaac tcgccaaatc actaagcatg tggcacaaat
3541 tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa
3601 agtgattacc ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa
3661 agtacgtgag attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg
3721 aactgctttg attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa
3781 agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc
3841 aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa
3901 tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg
3961 ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat
4021 tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag
4081 aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt
4141 tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc
4201 gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt
4261 tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt
4321 aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct
4381 ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa
4441 ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca
4501 aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag
4561 tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata
4621 taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac
4681 gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa
4741 acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg
4801 tctttatgaa acacgcattg atttgagtca gctaggaggt gactaactcg agtaaggatc
4861 tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
4921 tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc
4981 tgcgtttata cctagggaga cgtcggcgtt cggctgcggc gagcggtatc agctcactca
5041 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
5101 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
5161 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
5221 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
5281 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
5341 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
5401 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
5461 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
5521 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
5581 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
5641 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
5701 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
5761 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgactagt
5821 gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag
5881 atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc tcgtaaactt
5941 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
6001 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
6061 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
6121 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
6181 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
6241 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
6301 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
6361 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
6421 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
6481 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
6541 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
6601 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
6661 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
6721 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
6781 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
6841 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
6901 aaataggggt tccgcgcaca tttccccgaa aa
温馨提示：不可用于临床ZL。
