LOCUS       Exported                7753 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Gateway entry vector with pco-dCas9-VP64.
ACCESSION   .
VERSION     .
KEYWORDS    pYPQ152
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7753)
  AUTHORS   Lowder LG, Paul JW, Baltes NJ, Voytas DF, Zhang Y, Zhang D, Tang X, 
            Zheng X, Hsieh TF, Qi Y
  TITLE     A CRISPR/Cas9 toolbox for multiplexed plant genome editing and 
            transcriptional regulation.
  JOURNAL   Plant Physiol. 2015 Aug 21. pii: pp.00636.2015.
  PUBMED    26297141
REFERENCE   2  (bases 1 to 7753)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7753
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             359..1150
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1243..1831
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1732..1751
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1985..2002
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      2161..2188
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      2280..2366
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2415..2437
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2429..2446
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2430..2446
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2462..2561
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2583..2630
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             2637..2657
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2682..4118,4308..6971)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system"
                     /label=pcoCas9
                     /note="plant codon-optimized Cas9 gene containing the 
                     potato IV2 intron"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     intron          4119..4307
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     CDS             6972..7019
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      7204..7456
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    7501..7624
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7641..7660)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7642..7660)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7665..7681)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7665..7681)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctggaccagc caggacagaa atgcctcgac ttcgctgcta cccaaggttg ccgggtgacg
       61 cacaccgtgg aaacggatga aggcacgaac ccagtggaca taagcctgtt cggttcgtaa
      121 gctgtaatgc aagtagcgta tgcgctcacg caactggtcc agaaccttga ccgaacgcag
      181 cggtggtaac ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttggggtaca
      241 gtctatgcct cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt
      301 atggagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt taaacattat
      361 gagggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga
      421 gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg
      481 cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac
      541 aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga
      601 gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta
      661 tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat
      721 cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca
      781 tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga
      841 tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg
      901 cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa
      961 aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca
     1021 gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc
     1081 gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt
     1141 cggcaaataa ccctcgagcc acccatgacc aaaatccctt aacgtgagtt acgcgtcgtt
     1201 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1261 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1321 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     1381 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     1441 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     1501 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     1561 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     1621 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     1681 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     1741 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     1801 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     1861 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     1921 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     1981 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     2041 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     2101 cagtgagcgc aacgcaatta atacgcgtac cgcgagccag gaagagtttg tagaaacgca
     2161 aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg
     2221 cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga
     2281 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
     2341 tccgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct
     2401 tgcatggatg ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc
     2461 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca
     2521 atgctttttt ataatgccaa ctttgtacaa aaaagcaggc tccgaattcg cccttcacca
     2581 tggattacaa ggatgatgat gataaggatt acaaggatga tgatgataag atggctccaa
     2641 agaagaagag aaaggttgga atccacggag ttccagctgc tgataagaag tactctatcg
     2701 gacttgccat cggaaccaac tctgttggat gggctgttat caccgatgag tacaaggttc
     2761 catctaagaa gttcaaggtt cttggaaaca ccgatagaca ctctatcaag aagaacctta
     2821 tcggtgctct tcttttcgat tctggagaga ccgctgaggc taccagattg aagagaaccg
     2881 ctagaagaag atacaccaga agaaagaaca gaatctgcta ccttcaggaa atcttctcta
     2941 acgagatggc taaggttgat gattctttct tccacagact tgaggagtct ttccttgttg
     3001 aggaggataa gaagcacgag agacacccaa tcttcggaaa catcgttgat gaggttgctt
     3061 accacgagaa gtacccaacc atctaccacc ttagaaagaa gttggttgat tctaccgata
     3121 aggctgatct tagacttatc taccttgctc ttgctcacat gatcaagttc agaggacact
     3181 tccttatcga gggagacctt aacccagata actctgatgt tgataagttg ttcatccagc
     3241 ttgttcagac ctacaaccag cttttcgagg agaacccaat caacgcttct ggagttgatg
     3301 ctaaggctat cctttctgct agactttcta agtctcgtag acttgagaac cttatcgctc
     3361 agcttccagg agagaagaag aacggacttt tcggaaacct tatcgctctt tctcttggac
     3421 ttaccccaaa cttcaagtct aacttcgatc ttgctgagga tgctaagttg cagctttcta
     3481 aggataccta cgatgatgat cttgataacc ttcttgctca gatcggagat cagtacgctg
     3541 atcttttcct tgctgctaag aacctttctg atgctatcct tctttctgac atccttagag
     3601 ttaacaccga gatcaccaag gctccacttt ctgcttctat gatcaagaga tacgatgagc
     3661 accaccagga tcttaccctt ttgaaggctc ttgttagaca gcagcttcca gagaagtaca
     3721 aggaaatctt cttcgatcag tctaagaacg gatacgctgg atacatcgat ggaggagctt
     3781 ctcaggagga gttctacaag ttcatcaagc caatccttga gaagatggat ggaaccgagg
     3841 agcttcttgt taagttgaac agagaggatc ttcttagaaa gcagagaacc ttcgataacg
     3901 gatctatccc acaccagatc caccttggag agcttcacgc tatccttcgt agacaggagg
     3961 atttctaccc attcttgaag gataacagag agaagatcga gaagatcctt accttcagaa
     4021 tcccatacta cgttggacca cttgctagag gaaactctcg tttcgcttgg atgaccagaa
     4081 agtctgagga gaccatcacc ccttggaact tcgaggaggt aagtttctgc ttctaccttt
     4141 gatatatata taataattat cattaattag tagtaatata atatttcaaa tatttttttc
     4201 aaaataaaag aatgtagtat atagcaattg cttttctgta gtttataagt gtgtatattt
     4261 taatttataa cttttctaat atatgaccaa aatttgttga tgtgcaggtt gttgataagg
     4321 gagcttctgc tcagtctttc atcgagagaa tgaccaactt cgataagaac cttccaaacg
     4381 agaaggttct tccaaagcac tctcttcttt acgagtactt caccgtttac aacgagctta
     4441 ccaaggttaa gtacgttacc gagggaatga gaaagccagc tttcctttct ggagagcaga
     4501 agaaggctat cgttgatctt cttttcaaga ccaacagaaa ggttaccgtt aagcagttga
     4561 aggaggatta cttcaagaag atcgagtgct tcgattctgt tgaaatctct ggagttgagg
     4621 atagattcaa cgcttctctt ggaacctacc acgatctttt gaagatcatc aaggataagg
     4681 atttccttga taacgaggag aacgaggaca tccttgagga catcgttctt acccttaccc
     4741 ttttcgagga tagagagatg atcgaggaga gactcaagac ctacgctcac cttttcgatg
     4801 ataaggttat gaagcagttg aagagaagaa gatacaccgg atggggtaga ctttctcgta
     4861 agttgatcaa cggaatcaga gataagcagt ctggaaagac catccttgat ttcttgaagt
     4921 ctgatggatt cgctaacaga aacttcatgc agcttatcca cgatgattct cttaccttca
     4981 aggaggacat ccagaaggct caggtttctg gacagggaga ttctcttcac gagcacatcg
     5041 ctaaccttgc tggatctcca gctatcaaga agggaatcct tcagaccgtt aaggttgttg
     5101 atgagcttgt taaggttatg ggtagacaca agccagagaa catcgttatc gagatggcta
     5161 gagagaacca gaccacccag aagggacaga agaactctcg tgagagaatg aagagaatcg
     5221 aggagggaat caaggagctt ggatctcaaa tcttgaagga gcacccagtt gagaacaccc
     5281 agcttcagaa cgagaagttg tacctttact accttcagaa cggaagagat atgtacgttg
     5341 atcaggagct tgacatcaac agactttctg attacgatgt tgatgccatc gttccacagt
     5401 ctttcttgaa ggatgattct atcgataaca aggttcttac ccgttctgat aagaacagag
     5461 gaaagtctga taacgttcca tctgaggagg ttgttaagaa gatgaagaac tactggagac
     5521 agcttcttaa cgctaagttg atcacccaga gaaagttcga taaccttacc aaggctgaga
     5581 gaggaggact ttctgagctt gataaggctg gattcatcaa gagacagctt gttgagacca
     5641 gacagatcac caagcacgtt gctcagatcc ttgattctcg tatgaacacc aagtacgatg
     5701 agaacgataa gttgatcaga gaggttaagg ttatcacctt gaagtctaag ttggtttctg
     5761 atttcagaaa ggatttccag ttctacaagg ttagagagat caacaactac caccacgctc
     5821 acgatgctta ccttaacgct gttgttggaa ccgctcttat caagaagtac ccaaagttgg
     5881 agtctgagtt cgtttacgga gattacaagg tttacgatgt tagaaagatg atcgctaagt
     5941 ctgagcagga gatcggaaag gctaccgcta agtacttctt ctactctaac atcatgaact
     6001 tcttcaagac cgagatcacc cttgctaacg gagagatcag aaagagacca cttatcgaga
     6061 ccaacggaga gaccggagag atcgtttggg ataagggaag agatttcgct accgttagaa
     6121 aggttctttc tatgccacag gttaacatcg ttaagaaaac cgaggttcag accggaggat
     6181 tctctaagga gtctatcctt ccaaagagaa actctgataa gttgatcgct agaaagaagg
     6241 attgggaccc aaagaagtac ggaggattcg attctccaac cgttgcttac tctgttcttg
     6301 ttgttgctaa ggttgagaag ggaaagtcta agaagttgaa gtctgttaag gagcttcttg
     6361 gaatcaccat catggagcgt tcttctttcg agaagaaccc aatcgatttc cttgaggcta
     6421 agggatacaa ggaggttaag aaggatctta tcatcaagtt gccaaagtac tctcttttcg
     6481 agcttgagaa cggaagaaag agaatgcttg cttctgctgg agagcttcag aagggaaacg
     6541 agcttgctct tccatctaag tacgttaact tcctttacct tgcttctcac tacgagaagt
     6601 tgaagggatc tccagaggat aacgagcaga agcagctttt cgttgagcag cacaagcact
     6661 accttgatga gatcatcgag caaatctctg agttctctaa gagagttatc cttgctgatg
     6721 ctaaccttga taaggttctt tctgcttaca acaagcacag agataagcca atcagagagc
     6781 aggctgagaa catcatccac cttttcaccc ttaccaacct tggtgctcca gctgctttca
     6841 agtacttcga taccaccatc gatagaaaaa gatacacctc taccaaggag gttcttgatg
     6901 ctacccttat ccaccagtct atcaccggac tttacgagac cagaatcgat ctttctcagc
     6961 ttggaggaga taagagacca gctgctacca agaaggctgg acaggctaag aagaagaagg
     7021 gagacggctc tggatcgggg tcgggttctg gctcagtcga cgatgctctt gacgattttg
     7081 acctcgatat gctcgacgct cttgatgatt ttgatctcga catgctcgat gcacttgatg
     7141 actttgacct tgacatgctc gacgcactcg atgacttcga cctcgacatg ctttaggacg
     7201 tccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt
     7261 gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa
     7321 tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa
     7381 tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca
     7441 tctatgttac tagatcggga attgatcccc cctcgacagc ttccggaaag ggcgaattcg
     7501 caactttgta tacaaaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa
     7561 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac
     7621 tatgccatcc agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct
     7681 ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
     7741 catcatgcct cct
//
