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ptCC9 vector (V002803#)

Basic Vector Information

Basic Vector Information
Vector Name ptCC9 Antibiotic Resistance Ampicillin
Length 8857 bp Type Shuttle vector
Source Stukenberg D, Zauner S, Dell'Aquila G, Maier UG.

ptCC9 vector Vector Map

nitrate reductase cas9 nitrate reductase U6-RNA promoter guide RNA U6-RNA terminator fcpB promoter ble fcpB teminator T7 promoter ori beta-lactamase AmpR promoter

ptCC9 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                8857 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle vector ptCC9, complete sequence.
ACCESSION   MH143578
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8857)
  AUTHORS   Stukenberg D, Zauner S, Dell'Aquila G, Maier UG.
  TITLE     Optimizing CRISPR/Cas9 for the Diatom Phaeodactylum tricornutum
  JOURNAL   Front Plant Sci 9, 740 (2018)
  PUBMED    29928285
REFERENCE   2  (bases 1 to 8857)
  AUTHORS   Stuckenberg D, Zauner S.
  TITLE     Direct Submission
  JOURNAL   Submitted (29-MAR-2018) Philipps-University Marburg, Cellbiology, 
            Karl-von-Frisch Str. 8, Marburg 35043, Deutschland
REFERENCE   3  (bases 1 to 8857)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8857
                     /organism="Shuttle vector ptCC9"
                     /mol_type="other DNA"
                     /db_xref="taxon:2248355"
     regulatory      1..415
                     /regulatory_class="promoter"
                     /note="nitrate reductase; derived from Phaeodactylum 
                     tricornutum"
     CDS             416..4699
                     /codon_start=1
                     /gene="cas9"
                     /product="Cas9"
                     /label=cas9
                     /protein_id="AWY93773.1"
                     /translation="MASRMDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAD
                     KKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA
                     TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG
                     NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
                     DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF
                     GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS
                     DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK
                     NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH
                     LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETIT
                     PWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG
                     MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLG
                     TYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL
                     KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK
                     AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQT
                     TQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL
                     DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL
                     NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN
                     DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE
                     SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE
                     TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK
                     KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL
                     EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH
                     YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK
                     PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR
                     IDLSQLGGDKRPAATKKAGQAKKKK"
     gene            416..4699
                     /gene="cas9"
                     /label=cas9
                     /note="amplified from pKSdiaCas9_sgRNA (Nymark et al. 
                     (2016), obtained from AddGene; AddGene ID: 74923)"
     CDS             431..496
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             503..523
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             548..4648
                     /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             4649..4696
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     regulatory      4700..4972
                     /regulatory_class="terminator"
                     /note="nitrate reductase; derived from Phaeodactylum 
                     tricornutum"
     regulatory      5010..5288
                     /regulatory_class="promoter"
                     /note="U6-RNA promoter; derived from Phaeodactylum 
                     tricornutum"
     misc_RNA        5289..5384
                     /product="guide RNA"
     misc_RNA        5310..5385
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      5386..5723
                     /regulatory_class="terminator"
                     /note="U6-RNA terminator; derived from Phaeodactylum 
                     tricornutum"
     regulatory      5724..5916
                     /regulatory_class="promoter"
                     /note="fcpB promoter; derived from Phaeodactylum 
                     tricornutum"
     CDS             5917..6291
                     /codon_start=1
                     /gene="ble"
                     /product="Ble"
                     /label=ble
                     /note="zeocin resistance protein"
                     /protein_id="AWY93774.1"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     gene            5917..6291
                     /gene="ble"
                     /label=ble
     regulatory      6292..6532
                     /regulatory_class="terminator"
                     /note="fcpB teminator; derived from Phaeodactylum 
                     tricornutum"
     promoter        complement(6543..6561)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(6819..7407)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7578..8438)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /protein_id="AWY93775.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(8439..8543)
                     /gene="bla"
                     /label=AmpR promoter

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