CasYFP vector (V010040#)

Basic Vector Information

Basic Vector Information
Vector Name CasYFP Antibiotic Resistance Ampicillin
Length 10437 bp Type Cloning vector
Source Hahn F, Mantegazza O, Greiner A, Hegemann P, Eisenhut M, Weber AP.

CasYFP vector Vector Map

CasYFP10437 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000f1 oriSK primersgRNA scaffoldU6/C8from Chlamydomonas reinhardtiiCas9P2Aaminoglycoside 3'-phosphotransferase VIIIT3 promoterM13 revlac operatorlac promoterCAP binding siteColE1 originAmpRAmpR promoter

Plasmid Resuspension Protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

CasYFP vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported               10437 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector CasYFP, complete sequence.
ACCESSION   KY080688
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10437)
  AUTHORS   Hahn F, Mantegazza O, Greiner A, Hegemann P, Eisenhut M, Weber AP.
  TITLE     An Efficient Visual Screen for CRISPR/Cas9 Activity in Arabidopsis 
            thaliana
  JOURNAL   Front Plant Sci 8, 39 (2017)
  PUBMED    28174584
REFERENCE   2  (bases 1 to 10437)
  AUTHORS   Hahn F, Mantegazza O, Eisenhut M, Greiner A, Hegemann P, Weber APM.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-NOV-2016) Plant Biochemistry, Heinrich Heine 
            University Dusseldorf, Universitatsstr. 1, Dusseldorf 40225, Germany
REFERENCE   3  (bases 1 to 10437)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10437
                     /organism="Cloning vector CasYFP"
                     /mol_type="other DNA"
                     /db_xref="taxon:1955678"
     rep_origin      4..362
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(385..401)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(781..860)
                     /label=sgRNA scaffold
                     /note="sgRNA scaffold"
     misc_RNA        complement(785..860)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      complement(881..1380)
                     /regulatory_class="promoter"
                     /note="U6/C8"
     regulatory      1441..2254
                     /regulatory_class="promoter"
                     /note="from Chlamydomonas reinhardtii"
     CDS             2258..7114
                     /codon_start=1
                     /gene="Cas9"
                     /product="NLS-Cas9-eYFP"
                     /label=Cas9
                     /note="Cas9 with nuclear localization signal and eYFP; 
                     Chlamydomonas reinhardtii codon optimized Cas9"
                     /protein_id="AQN67804.1"
                     /translation="MASSPPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKV
                     LGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKV
                     DDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR
                     LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI
                     LSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT
                     YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH
                     QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE
                     LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFR
                     IPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK
                     VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK
                     EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT
                     LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL
                     KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVK
                     VVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV
                     ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS
                     DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR
                     QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI
                     NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF
                     FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK
                     KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSK
                     KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML
                     ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQI
                     SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID
                     RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDTGVSKGEELFTGVVPILVELDGD
                     VNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQ
                     HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH
                     KLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN
                     HYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     gene            2258..7114
                     /gene="Cas9"
                     /label=Cas9
     CDS             2273..2293
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2294..6394
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7130..7186
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7187..7987
                     /codon_start=1
                     /product="aminoglycoside 3'-phosphotransferase VIII"
                     /function="Selection marker in Chlamydomonas reinhardtii"
                     /label=aminoglycoside 3'-phosphotransferase VIII
                     /note="selection marker aminoglycoside 
                     3'-phosphotransferase gene (aph) from Streptomyces rimosus"
                     /protein_id="AQN67803.1"
                     /translation="DDALRALRGRYPGCEWVVVEDGASGAGVYRLRGGGRELFVKVAAL
                     GAGVGLLGEAERLVWLAEVGIPVPRVVEGGGDERVAWLVTEAVPGRPASARWPREQRLD
                     VAVALAGLARSLHALDWERCPFDRSLAVTVPQAARAVAEGSVDLEDLDEERKGWSGERL
                     LAELERTRPADEDLAVCHGDLCPDNVLLDPRTCEVTGLIDVGRVGRADRHSDLALVLRE
                     LAHEEDPWFGPECSAAFLREYGRGWDGAVSEEKLAFYRLLDEFF"
     regulatory      7991..8230
                     /regulatory_class="terminator"
     promoter        complement(8249..8267)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8288..8304)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8312..8328
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(8336..8366)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8381..8402
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      8672..9300
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      complement(8690..9278)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9449..10309)
                     /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"
     CDS             complement(9449..10111)
                     /codon_start=1
                     /product="ampicillin resistance protein"
                     /label=ampicillin resistance protein
                     /protein_id="AQN67805.1"
                     /translation="MSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHL
                     TDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEA
                     IPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFI
                     ADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     promoter        complement(10310..10414)
                     /gene="bla"
                     /label=AmpR promoter
     regulatory      complement(10351..10379)
                     /regulatory_class="promoter"

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