LOCUS       sequence-198621-        7579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pNB184-Cas9 entry cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7579)
  AUTHORS   Curtin SJ, Xiong Y, Michno JM, Campbell BW, Stec AO, Cermak T, 
            Starker C, Voytas DF, Eamens AL, Stupar RM
  TITLE     CRISPR/Cas9 and TALENs generate heritable mutations for genes 
            involved in small RNA processing of Glycine max and Medicago 
            truncatula.
  JOURNAL   Plant Biotechnol J. 2017 Oct 31. doi: 10.1111/pbi.12857.
  PUBMED    29087011
REFERENCE   2  (bases 1 to 7579)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Biotechnol J. 2017 Oct 31. doi: 10.1111/pbi.12857."
COMMENT     Sequence Label: pSC12
FEATURES             Location/Qualifiers
     source          1..7579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      8..260
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    305..428
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(445..464)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(446..464)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(469..485)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(469..485)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             916..1707
                     /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      1800..2388
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2289..2308
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2542..2559
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2718..2745)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2837..2923)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2972..2994
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2986..3003
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2987..3003
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3019..3118
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3140..3187
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             3194..3214
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3239..4675,4865..7528)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     intron          4676..4864
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     CDS             7529..7576
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 gacgtccgat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg
       61 tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat
      121 gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat
      181 ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt
      241 gtcatctatg ttactagatc gggaattgat cccccctcga cagcttccgg aaagggcgaa
      301 ttcgcaactt tgtatacaaa agttgaacga gaaacgtaaa atgatataaa tatcaatata
      361 ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag
      421 tcactatgcc atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt
      481 tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat
      541 atatcatcat gcctcctctg gaccagccag gacagaaatg cctcgacttc gctgctaccc
      601 aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca gtggacataa
      661 gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga
      721 accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac
      781 tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
      841 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
      901 acaaagttaa acatcatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
      961 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
     1021 tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc
     1081 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
     1141 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
     1201 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
     1261 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
     1321 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
     1381 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
     1441 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     1501 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     1561 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     1621 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc
     1681 gagatcacca aggtagtcgg caaataaccc tcgagccacc catgaccaaa atcccttaac
     1741 gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1801 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1861 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1921 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1981 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2041 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2101 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2161 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2221 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2281 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2341 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2401 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2461 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     2521 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     2581 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     2641 cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc gagccaggaa
     2701 gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc
     2761 tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca
     2821 aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa
     2881 aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct
     2941 actctcgcgt taacgcttgc atggatgttt tcccagtcac gacgttgtaa aacgacggcc
     3001 agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac ctgttcgttg
     3061 caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa agcaggctcc
     3121 gaattcgccc ttcaccatgg attacaagga tgatgatgat aaggattaca aggatgatga
     3181 tgataagatg gctccaaaga agaagagaaa ggttggaatc cacggagttc cagctgctga
     3241 taagaagtac tctatcggac ttgacatcgg aaccaactct gttggatggg ctgttatcac
     3301 cgatgagtac aaggttccat ctaagaagtt caaggttctt ggaaacaccg atagacactc
     3361 tatcaagaag aaccttatcg gtgctcttct tttcgattct ggagagaccg ctgaggctac
     3421 cagattgaag agaaccgcta gaagaagata caccagaaga aagaacagaa tctgctacct
     3481 tcaggaaatc ttctctaacg agatggctaa ggttgatgat tctttcttcc acagacttga
     3541 ggagtctttc cttgttgagg aggataagaa gcacgagaga cacccaatct tcggaaacat
     3601 cgttgatgag gttgcttacc acgagaagta cccaaccatc taccacctta gaaagaagtt
     3661 ggttgattct accgataagg ctgatcttag acttatctac cttgctcttg ctcacatgat
     3721 caagttcaga ggacacttcc ttatcgaggg agaccttaac ccagataact ctgatgttga
     3781 taagttgttc atccagcttg ttcagaccta caaccagctt ttcgaggaga acccaatcaa
     3841 cgcttctgga gttgatgcta aggctatcct ttctgctaga ctttctaagt ctcgtagact
     3901 tgagaacctt atcgctcagc ttccaggaga gaagaagaac ggacttttcg gaaaccttat
     3961 cgctctttct cttggactta ccccaaactt caagtctaac ttcgatcttg ctgaggatgc
     4021 taagttgcag ctttctaagg atacctacga tgatgatctt gataaccttc ttgctcagat
     4081 cggagatcag tacgctgatc ttttccttgc tgctaagaac ctttctgatg ctatccttct
     4141 ttctgacatc cttagagtta acaccgagat caccaaggct ccactttctg cttctatgat
     4201 caagagatac gatgagcacc accaggatct tacccttttg aaggctcttg ttagacagca
     4261 gcttccagag aagtacaagg aaatcttctt cgatcagtct aagaacggat acgctggata
     4321 catcgatgga ggagcttctc aggaggagtt ctacaagttc atcaagccaa tccttgagaa
     4381 gatggatgga accgaggagc ttcttgttaa gttgaacaga gaggatcttc ttagaaagca
     4441 gagaaccttc gataacggat ctatcccaca ccagatccac cttggagagc ttcacgctat
     4501 ccttcgtaga caggaggatt tctacccatt cttgaaggat aacagagaga agatcgagaa
     4561 gatccttacc ttcagaatcc catactacgt tggaccactt gctagaggaa actctcgttt
     4621 cgcttggatg accagaaagt ctgaggagac catcacccct tggaacttcg aggaggtaag
     4681 tttctgcttc tacctttgat atatatataa taattatcat taattagtag taatataata
     4741 tttcaaatat ttttttcaaa ataaaagaat gtagtatata gcaattgctt ttctgtagtt
     4801 tataagtgtg tatattttaa tttataactt ttctaatata tgaccaaaat ttgttgatgt
     4861 gcaggttgtt gataagggag cttctgctca gtctttcatc gagagaatga ccaacttcga
     4921 taagaacctt ccaaacgaga aggttcttcc aaagcactct cttctttacg agtacttcac
     4981 cgtttacaac gagcttacca aggttaagta cgttaccgag ggaatgagaa agccagcttt
     5041 cctttctgga gagcagaaga aggctatcgt tgatcttctt ttcaagacca acagaaaggt
     5101 taccgttaag cagttgaagg aggattactt caagaagatc gagtgcttcg attctgttga
     5161 aatctctgga gttgaggata gattcaacgc ttctcttgga acctaccacg atcttttgaa
     5221 gatcatcaag gataaggatt tccttgataa cgaggagaac gaggacatcc ttgaggacat
     5281 cgttcttacc cttacccttt tcgaggatag agagatgatc gaggagagac tcaagaccta
     5341 cgctcacctt ttcgatgata aggttatgaa gcagttgaag agaagaagat acaccggatg
     5401 gggtagactt tctcgtaagt tgatcaacgg aatcagagat aagcagtctg gaaagaccat
     5461 ccttgatttc ttgaagtctg atggattcgc taacagaaac ttcatgcagc ttatccacga
     5521 tgattctctt accttcaagg aggacatcca gaaggctcag gtttctggac agggagattc
     5581 tcttcacgag cacatcgcta accttgctgg atctccagct atcaagaagg gaatccttca
     5641 gaccgttaag gttgttgatg agcttgttaa ggttatgggt agacacaagc cagagaacat
     5701 cgttatcgag atggctagag agaaccagac cacccagaag ggacagaaga actctcgtga
     5761 gagaatgaag agaatcgagg agggaatcaa ggagcttgga tctcaaatct tgaaggagca
     5821 cccagttgag aacacccagc ttcagaacga gaagttgtac ctttactacc ttcagaacgg
     5881 aagagatatg tacgttgatc aggagcttga catcaacaga ctttctgatt acgatgttga
     5941 tcacatcgtt ccacagtctt tcttgaagga tgattctatc gataacaagg ttcttacccg
     6001 ttctgataag aacagaggaa agtctgataa cgttccatct gaggaggttg ttaagaagat
     6061 gaagaactac tggagacagc ttcttaacgc taagttgatc acccagagaa agttcgataa
     6121 ccttaccaag gctgagagag gaggactttc tgagcttgat aaggctggat tcatcaagag
     6181 acagcttgtt gagaccagac agatcaccaa gcacgttgct cagatccttg attctcgtat
     6241 gaacaccaag tacgatgaga acgataagtt gatcagagag gttaaggtta tcaccttgaa
     6301 gtctaagttg gtttctgatt tcagaaagga tttccagttc tacaaggtta gagagatcaa
     6361 caactaccac cacgctcacg atgcttacct taacgctgtt gttggaaccg ctcttatcaa
     6421 gaagtaccca aagttggagt ctgagttcgt ttacggagat tacaaggttt acgatgttag
     6481 aaagatgatc gctaagtctg agcaggagat cggaaaggct accgctaagt acttcttcta
     6541 ctctaacatc atgaacttct tcaagaccga gatcaccctt gctaacggag agatcagaaa
     6601 gagaccactt atcgagacca acggagagac cggagagatc gtttgggata agggaagaga
     6661 tttcgctacc gttagaaagg ttctttctat gccacaggtt aacatcgtta agaaaaccga
     6721 ggttcagacc ggaggattct ctaaggagtc tatccttcca aagagaaact ctgataagtt
     6781 gatcgctaga aagaaggatt gggacccaaa gaagtacgga ggattcgatt ctccaaccgt
     6841 tgcttactct gttcttgttg ttgctaaggt tgagaaggga aagtctaaga agttgaagtc
     6901 tgttaaggag cttcttggaa tcaccatcat ggagcgttct tctttcgaga agaacccaat
     6961 cgatttcctt gaggctaagg gatacaagga ggttaagaag gatcttatca tcaagttgcc
     7021 aaagtactct cttttcgagc ttgagaacgg aagaaagaga atgcttgctt ctgctggaga
     7081 gcttcagaag ggaaacgagc ttgctcttcc atctaagtac gttaacttcc tttaccttgc
     7141 ttctcactac gagaagttga agggatctcc agaggataac gagcagaagc agcttttcgt
     7201 tgagcagcac aagcactacc ttgatgagat catcgagcaa atctctgagt tctctaagag
     7261 agttatcctt gctgatgcta accttgataa ggttctttct gcttacaaca agcacagaga
     7321 taagccaatc agagagcagg ctgagaacat catccacctt ttcaccctta ccaaccttgg
     7381 tgctccagct gctttcaagt acttcgatac caccatcgat agaaaaagat acacctctac
     7441 caaggaggtt cttgatgcta cccttatcca ccagtctatc accggacttt acgagaccag
     7501 aatcgatctt tctcagcttg gaggagataa gagaccagct gctaccaaga aggctggaca
     7561 ggctaagaag aagaagtga
//
