LOCUS       40924_25316        6551 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses an sgRNA targeting the PDS gene in Nicotiana benthamiana 
            from the Arabidopsis U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6551)
  AUTHORS   Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S
  TITLE     Targeted mutagenesis in the model plant Nicotiana benthamiana using 
            Cas9 RNA-guided endonuclease.
  JOURNAL   Nat Biotechnol. 2013 Aug 8;31(8):691-3. doi: 10.1038/nbt.2655.
  PUBMED    23929340
REFERENCE   2  (bases 1 to 6551)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6551)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt"; 
            journalName: "Nat Biotechnol"; date: "2013-08-8- 8"; volume: "31"; 
            issue: "8"; pages: "691-3"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    32..56
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(179..767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(941..1732)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MAKMRISPELKKLIEKYRCAKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        complement(1733..1837)
                     /label=AmpR promoter
     rep_origin      complement(2120..2314)
                     /direction=LEFT
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(2383..3453)
                     /codon_start=1
                     /label=pVS1 RepA
                     /note="replication protein from the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
                     /translation="VSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGASSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     CDS             complement(3885..4511)
                     /codon_start=1
                     /label=pVS1 StaA
                     /note="stability protein from the Pseudomonas plasmid pVS1
                     (Heeb et al., 2000)"
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     misc_feature    4775..4799
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        5009..5192
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     CDS             5213..6004
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     terminator      6033..6285
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        6312..6386
                     /label=AtU6 promoter
                     /note="consensus promoter from the three Arabidopsis U6
                     snRNA genes (Waibel and Filipowicz, 1990; Nekrasov et al., 
                     2013)"
     misc_RNA        6407..6482
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(6494..6518)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 gggacacgaa gtggtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa
       61 gagaaaagag cgtttattag aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg
      121 ttcgtccatt tgtatgtgcc aaccacagga accgtaaaaa ggccgcgttg ctggcgtttt
      181 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      241 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
      301 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
      361 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
      421 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
      481 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
      541 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
      601 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
      661 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
      721 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
      781 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
      841 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
      901 caatctaaag tatatatgag taaacttggt ctgacagcta aaacaattca tccagtaaaa
      961 tataatattt tattttctcc caatcaggct tgatccccag taagtcaaaa aatagctcga
     1021 catactgttc ttccccgata tcctccctga tcgaccggac gcagaaggca atgtcatacc
     1081 acttgtccgc cctgccgctt ctcccaagat caataaagcc acttactttg ccatctttca
     1141 caaagatgtt gctgtctccc aggtcgccgt gggaaaagac aagttcctct tcgggctttt
     1201 ccgtctttaa aaaatcatac agctcgcgcg gatctttaaa tggagtgtcc tcttcccagt
     1261 tttcgcaatc cacatcggcc agatcgttat tcagtaagta atccaattcg gctaagcggc
     1321 tgtctaagct attcgtatag ggacaatccg atatgtcgat ggagtgaaag agcctgatgc
     1381 actccgcata cagctcgata atcttttcag ggctttgttc atcttcatac tcttccgagc
     1441 aaaggacgcc atcggcctca ctcatgagca gattgctcca gccatcatgc cgttcaaagt
     1501 gcaggacctt tggaacaggc agctttcctt ccagccatag catcatgtcc ttttcccgtt
     1561 ccacatcata ggtggtccct ttataccggc tgtccgtcat ttttaaatat aggttttcat
     1621 tttctcccac cagcttatat accttagcag gagacattcc ttccgtatct tttgcgcagc
     1681 ggtatttttc gatcagtttt ttcaattccg gtgatattct cattttagcc atactcttcc
     1741 tttttcaata ttattgaagc atttatcaag gttattgtct catgagcgga tacatatttg
     1801 aatgtattta gaaaaataaa caaatagggg ttccgcgcac gaattggcca gcgctgccat
     1861 ttttggggtg aggccgttcg cggccgaggg gcgcagcccc tggggggatg ggagctcggt
     1921 gccttgatgt gggcgccggc ggtcgagtgg cgacggcgcg gcttgtccgc gccctggtag
     1981 attgcctggc cgtaggccag ccatttttga gcggccagcg gccgcgatag gccgacgcga
     2041 agcggcgggg cgtagggagc gcagcgaccg aagggtaggc gctttttgca gctcttcggc
     2101 tgtgcgctgg ccagacagtt atgcacaggc caggcgggtt ttaagagttt taataagttt
     2161 taaagagttt taggcggaaa aatcgccttt tttctctttt atatcagtca cttacatgtg
     2221 tgaccggttc ccaatgtacg gctttgggtt cccaatgtac gggttccggt tcccaatgta
     2281 cggctttggg ttcccaatgt acgtgctatc cacaggaaag aagctttttc gacctttttc
     2341 ccctgctagg gcaatttgcc ctagcatctg ctccgtacat taggaaccgg cggatgcttc
     2401 gccctcgatc aggttgcggt agcgcatgac taggatcggg ccagcctgcc ccgcctcctc
     2461 cttcaaatcg tactccggca ggtcatttga cccgatcagc ttgcgcacgg tgaaacagaa
     2521 cttcttgaac tctccggcgc tgccactgcg ttcgtagatc gtcttgaaca accatctggc
     2581 ttctgccttg cctgcggcgc ggcgtgccag gcggtagaga aaacggccga tgccgggatc
     2641 gatcaaaaag taatcggggt gaaccgtcag cacgtccggg ttcttgcctt ctgtgatctc
     2701 gcggtacatc caatcagcta gctcgatctc gatgtactcc ggccgcccgg tttcgctctt
     2761 tacgatcttg tagcggctaa tcaaggcttc accctcggat accgtcacca ggcggccgtt
     2821 cttggccttc ttcgtacgct gcatggcaac gtgcgtggtg tttaaccgaa tgcaggtttc
     2881 taccaggtcg tctttctgct ttccgccatc ggctcgccgg cagaacttga gtacgtccgc
     2941 aacgtgtgga cggaacacgc ggccgggctt gtctcccttc ccttcccggt atcggttcat
     3001 ggattcggtt agatgggaaa ccgccatcag taccaggtcg taatcccaca cactggccat
     3061 gccggccggc cctgcggaaa cctctacgtg cccgtctgga agctcgtagc ggatcacctc
     3121 gccagctcgt cggtcacgct tcgacagacg gaaaacggcc acgtccatga tgctgcgact
     3181 atcgcgggtg cccacgtcat agagcatcgg aacgaaaaaa tctggttgct cgtcgccctt
     3241 gggcggcttc ctaatcgacg acgcaccggc tgccggcggt tgccgggatt ctttgcggat
     3301 tcgatcagcg gccgcttgcc acgattcacc ggggcgtgct tctgcctcga tgcgttgccg
     3361 ctgggcggcc tgcgcggcct tcaacttctc caccaggtca tcacccagcg ccgcgccgat
     3421 ttgtaccggg ccggatggtt tgcgaccgct cacgccgatt cctcgggctt gggggttcca
     3481 gtgccattgc agggccggca gacaacccag ccgcttacgc ctggccaacc gcccgttcct
     3541 ccacacatgg ggcattccac ggcgtcggtg cctggttgtt cttgattttc catgccgcct
     3601 cctttagccg ctaaaattca tctactcatt tattcatttg ctcatttact ctggtagctg
     3661 cgcgatgtat tcagatagca gctcggtaat ggtcttgcct tggcgtaccg cgtacatctt
     3721 cagcttggtg tgatcctccg ccggcaactg aaagttgacc cgcttcatgg ctggcgtgtc
     3781 tgccaggctg gccaacgttg cagccttgct gctgcgtgcg ctcggacggc cggcacttag
     3841 cgtgtttgtg cttttgctca ttttctcttt acctcattaa ctcaaatgag ttttgattta
     3901 atttcagcgg ccagcgcctg gacctcgcgg gcagcgtcgc cctcgggttc tgattcaaga
     3961 acggttgtgc cggcggcggc agtgcctggg tagctcacgc gctgcgtgat acgggactca
     4021 agaatgggca gctcgtaccc ggccagcgcc tcggcaacct caccgccgat gcgcgtgcct
     4081 ttgatcgccc gcgacacgac aaaggccgct tgtagccttc catccgtgac ctcaatgcgc
     4141 tgcttaacca gctccaccag gtcggcggtg gcccatatgt cgtaagggct tggctgcacc
     4201 ggaatcagca cgaagtcggc tgccttgatc gcggacacag ccaagtccgc cgcctggggc
     4261 gctccgtcga tcactacgaa gtcgcgccgg ccgatggcct tcacgtcgcg gtcaatcgtc
     4321 gggcggtcga tgccgacaac ggttagcggt tgatcttccc gcacggccgc ccaatcgcgg
     4381 gcactgccct ggggatcgga atcgactaac agaacatcgg ccccggcgag ttgcagggcg
     4441 cgggctagat gggttgcgat ggtcgtcttg cctgacccgc ctttctggtt aagtacagcg
     4501 ataaccttca tgcgttcccc ttgcgtattt gtttatttac tcatcgcatc atatacgcag
     4561 cgaccgcatg acgcaagctg ttttactcaa atacacatca cctttttaga cggcggcgct
     4621 cggtttcttc agcggccaag ctggccggcc aggccgccag cttggcatca gacaaaccgg
     4681 ccaggatttc atgcagccgc acggctgatg ggctgcctgt atcgagtggt gattttgtgc
     4741 cgagctgccg gtcggggagc tgttggctgg ctggtggcag gatatattgt ggtgtaaaca
     4801 aattgacgct tagacaactt aataacacat tgcggacgtt tttaatgtac tggggtggat
     4861 gcagtgggcc ccactctgtg tgccgaattc ggatccggag agcggagaat taagggagtc
     4921 acgttatgac ccccgccgat gacgcgggac aagccgtttt acgtttggaa ctgacagaac
     4981 cgcaacgttg aaggagccac tcagccgcgg gtttctggag tttaatgagc taagcacata
     5041 cgtcagaaac cattattgcg cgttcaaaag tcgcctaagg tcactatcag ctagcaaata
     5101 tttcttgtca aaaatgctcc actgacgttc cataaattcc cctcggtatc caattagagt
     5161 ctcatattca ctctcaatcc aaataatctg caccggatct ggatcgtttc gcatgattga
     5221 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     5281 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     5341 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tccaggacga
     5401 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     5461 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     5521 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     5581 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     5641 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     5701 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgtatgcccg acggcgagga
     5761 tctcgtcgtg actcatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     5821 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     5881 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     5941 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     6001 cttctgagcg ggtctggatc tagagtcaag cagatcgttc aaacatttgg caataaagtt
     6061 tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt
     6121 acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta
     6181 tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa
     6241 actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgacgc tgcaagaatt
     6301 caagcttgga gtgatcaaaa gtcccacatc gatcaggtga tatatagcag cttagtttat
     6361 ataatgatag agtcgacata gcgattgccg ttaatttgag agtccagttt tagagctaga
     6421 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     6481 gctttttttc tagacccagc tttcttgtac aaagttggca ttacgctact agaggatgca
     6541 catgtgaccg a
//
