| 载体基本信息 | |||
| 载体名称 | eSpCas9(1.1) | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 8506 bp | 载体类型 | CRISPR Plasmids |
| 载体来源 | Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. | 拷贝数 | High copy number |
eSpCas9(1.1) 载体载体图谱
eSpCas9(1.1) 载体载体序列
LOCUS Exported 8506 bp ds-DNA circular SYN 03-2-2016
DEFINITION Zhang lab vector for expressing a single guide RNA (sgRNA) together
with an S. pyogenes Cas9 variant with improved targeting
specificity.
ACCESSION .
VERSION .
KEYWORDS eSpCas9(1.1)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8506)
AUTHORS Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F.
TITLE Rationally engineered Cas9 nucleases with improved specificity.
JOURNAL Science 2016;351:84-8.
PUBMED 26628643
REFERENCE 2 (bases 1 to 8506)
AUTHORS Zhang Lab / Addgene #71814
TITLE Direct Submission
COMMENT An error in codon 848 was corrected based on sequencing results from
Addgene.
FEATURES Location/Qualifiers
source 1..8506
/organism="synthetic DNA construct"
/lab_host="Mammalian Cells"
/mol_type="other DNA"
promoter 1..241
/note="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 268..343
/note="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator 344..349
/note="polIII terminator"
/note="RNA polymerase III transcription terminator"
enhancer 440..725
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 727..1004
/note="chicken beta-actin promoter"
intron 1005..1233
/note="hybrid intron"
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 1251..1253
/codon_start=1
/product="start codon"
/note="ATG"
/translation="M"
CDS 1254..1319
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1326..1346
/codon_start=1
/locus_tag="
"
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/note="
"
/protein_id="
"
/translation="PKKKRKV"
CDS 1371..5471
/codon_start=1
/product="Cas9 endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system, mutated to improve targeting
specificity (Slaymaker et al., 2016)"
/note="eSpCas9(1.1)"
/note="carries the mutations K848A, K1003A, and R1060A"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5472..5519
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
polyA_signal 5553..5760
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
repeat_region 5769..5909
/note="AAV2 ITR"
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 5984..6439
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6721..6825
/gene="bla"
/note="AmpR promoter"
CDS 6826..7686
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 7857..8445
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
我们不提供该载体，载体信息仅供查询和参考。