LOCUS       Exported               10749 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pLX-B2-XT1-XT2-hCas9, complete sequence.
ACCESSION   KY825143
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10749)
  AUTHORS   Pasin F, Bedoya LC, Bernabe-Orts JM, Gallo A, Simon-Mateo C, Orzaez 
            D, Garcia JA.
  TITLE     Multiple T-DNA Delivery to Plants Using Novel Mini Binary Vectors 
            with Compatible Replication Origins
  JOURNAL   ACS Synth Biol (2017) In press
  PUBMED    28657330
REFERENCE   2  (bases 1 to 10749)
  AUTHORS   Pasin F, Garcia JA.
  TITLE     Direct Submission
  JOURNAL   Submitted (26-MAR-2017) Department of Plant Molecular Genetics, 
            Centro Nacional de Biotecnologia (CNB-CSIC), Darwin 3, Madrid, 
            Madrid 28049, Spain
REFERENCE   3  (bases 1 to 10749)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10749
                     /organism="Cloning vector pLX-B2-XT1-XT2-hCas9"
                     /mol_type="other DNA"
                     /db_xref="taxon:2016264"
     misc_feature    1..24
                     /label=assembly linker
                     /note="assembly linker"
     regulatory      25..72
                     /regulatory_class="terminator"
     misc_feature    133..157
                     /label=LB
                     /note="LB"
     misc_feature    180..210
                     /label=LB
                     /note="LB"
     misc_feature    184..208
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     regulatory      complement(267..581)
                     /regulatory_class="terminator"
                     /note="nos"
     terminator      287..539
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(579..1373)
                     /codon_start=1
                     /product="NptII"
                     /label=NptII
                     /note="kanamycin resistance protein"
                     /protein_id="ASG92121.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     regulatory      complement(1374..1664)
                     /regulatory_class="promoter"
                     /note="nos"
     promoter        complement(1407..1586)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     regulatory      1677..1878
                     /regulatory_class="promoter"
                     /note="U626 (Arabidopsis thaliana)"
     misc_RNA        1902..1984
                     /label=sgRNA
                     /note="sgRNA"
     misc_RNA        1902..1977
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1989..2013)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2031..2232
                     /regulatory_class="promoter"
                     /note="U626 (Arabidopsis thaliana)"
     misc_RNA        2256..2338
                     /label=sgRNA
                     /note="sgRNA"
     misc_RNA        2256..2331
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2343..2367)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2385..3411
                     /regulatory_class="promoter"
                     /note="2x CaMV 35S"
     promoter        2996..3340
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             3413..7552
                     /codon_start=1
                     /product="hCas9"
                     /label=hCas9
                     /protein_id="ASG92122.1"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGDSRADPKKKRKV"
     CDS             3413..7516
                     /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"
     misc_feature    7529..7549
                     /label=SV40 NLS
                     /note="SV40 NLS"
     CDS             7529..7549
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      7553..8048
                     /regulatory_class="terminator"
                     /note="nos"
     terminator      7576..7828
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    8115..8138
                     /label=RB
                     /note="RB"
     regulatory      complement(8203..8243)
                     /regulatory_class="terminator"
     misc_feature    8253..8276
                     /label=assembly linker
                     /note="assembly linker"
     CDS             complement(8287..9102)
                     /codon_start=1
                     /gene="neo"
                     /product="neomycin phosphotransferase"
                     /label=neo
                     /note="kanamycin resistance protein; NptI"
                     /protein_id="ASG92123.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(8287..9102)
                     /gene="neo"
                     /label=neo
     misc_feature    9204..9227
                     /label=assembly linker
                     /note="assembly linker"
     CDS             complement(9228..9890)
                     /codon_start=1
                     /gene="rep"
                     /product="replication protein"
                     /label=rep
                     /protein_id="ASG92124.1"
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     gene            complement(9228..9890)
                     /gene="rep"
                     /label=rep
     rep_origin      9891..10662
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"