LOCUS       sequence-202650-        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      6..258
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    303..426
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(443..462)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(444..462)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(467..483)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(467..483)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             914..1705
                     /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      1798..2386
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2287..2306
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2540..2557
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2716..2743)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2835..2921)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2970..2992
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2984..3001
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2985..3001
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3017..3116
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3138..3185
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             3192..3212
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3237..4673,4863..7526)
                     /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          4674..4862
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     CDS             7527..7574
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 cgtccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc
       61 ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt
      121 aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt
      181 aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt
      241 catctatgtt actagatcgg gaattgatcc cccctcgaca gcttccggaa agggcgaatt
      301 cgcaactttg tatacaaaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt
      361 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc
      421 actatgccat ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc
      481 ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
      541 atcatcatgc ctcctctgga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa
      601 ggttgccggg tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc
      661 ctgttcggtt cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac
      721 cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg
      781 tttttttggg gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg
      841 tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac
      901 aaagttaaac attatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt
      961 agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc
     1021 cgcagtggat ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt
     1081 aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc
     1141 ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat
     1201 cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga
     1261 cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac
     1321 aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc
     1381 ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc
     1441 gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag
     1501 cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct
     1561 gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga
     1621 agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga
     1681 gatcaccaag gtagtcggca aataaccctc gagccaccca tgaccaaaat cccttaacgt
     1741 gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1801 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1861 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1921 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1981 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2041 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     2101 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     2161 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2221 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2281 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2341 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2401 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2461 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2521 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     2581 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     2641 cgactggaaa gcgggcagtg agcgcaacgc aattaatacg cgtaccgcga gccaggaaga
     2701 gtttgtagaa acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg
     2761 gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa
     2821 tccgctcccg gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa
     2881 cgaaaggccc agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac
     2941 tctcgcgtta acgcttgcat ggatgttttc ccagtcacga cgttgtaaaa cgacggccag
     3001 tcttaagctc gggccccaaa taatgatttt attttgactg atagtgacct gttcgttgca
     3061 acaaattgat gagcaatgct tttttataat gccaactttg tacaaaaaag caggctccga
     3121 attcgccctt caccatggat tacaaggatg atgatgataa ggattacaag gatgatgatg
     3181 ataagatggc tccaaagaag aagagaaagg ttggaatcca cggagttcca gctgctgata
     3241 agaagtactc tatcggactt gacatcggaa ccaactctgt tggatgggct gttatcaccg
     3301 atgagtacaa ggttccatct aagaagttca aggttcttgg aaacaccgat agacactcta
     3361 tcaagaagaa ccttatcggt gctcttcttt tcgattctgg agagaccgct gaggctacca
     3421 gattgaagag aaccgctaga agaagataca ccagaagaaa gaacagaatc tgctaccttc
     3481 aggaaatctt ctctaacgag atggctaagg ttgatgattc tttcttccac agacttgagg
     3541 agtctttcct tgttgaggag gataagaagc acgagagaca cccaatcttc ggaaacatcg
     3601 ttgatgaggt tgcttaccac gagaagtacc caaccatcta ccaccttaga aagaagttgg
     3661 ttgattctac cgataaggct gatcttagac ttatctacct tgctcttgct cacatgatca
     3721 agttcagagg acacttcctt atcgagggag accttaaccc agataactct gatgttgata
     3781 agttgttcat ccagcttgtt cagacctaca accagctttt cgaggagaac ccaatcaacg
     3841 cttctggagt tgatgctaag gctatccttt ctgctagact ttctaagtct cgtagacttg
     3901 agaaccttat cgctcagctt ccaggagaga agaagaacgg acttttcgga aaccttatcg
     3961 ctctttctct tggacttacc ccaaacttca agtctaactt cgatcttgct gaggatgcta
     4021 agttgcagct ttctaaggat acctacgatg atgatcttga taaccttctt gctcagatcg
     4081 gagatcagta cgctgatctt ttccttgctg ctaagaacct ttctgatgct atccttcttt
     4141 ctgacatcct tagagttaac accgagatca ccaaggctcc actttctgct tctatgatca
     4201 agagatacga tgagcaccac caggatctta cccttttgaa ggctcttgtt agacagcagc
     4261 ttccagagaa gtacaaggaa atcttcttcg atcagtctaa gaacggatac gctggataca
     4321 tcgatggagg agcttctcag gaggagttct acaagttcat caagccaatc cttgagaaga
     4381 tggatggaac cgaggagctt cttgttaagt tgaacagaga ggatcttctt agaaagcaga
     4441 gaaccttcga taacggatct atcccacacc agatccacct tggagagctt cacgctatcc
     4501 ttcgtagaca ggaggatttc tacccattct tgaaggataa cagagagaag atcgagaaga
     4561 tccttacctt cagaatccca tactacgttg gaccacttgc tagaggaaac tctcgtttcg
     4621 cttggatgac cagaaagtct gaggagacca tcaccccttg gaacttcgag gaggtaagtt
     4681 tctgcttcta cctttgatat atatataata attatcatta attagtagta atataatatt
     4741 tcaaatattt ttttcaaaat aaaagaatgt agtatatagc aattgctttt ctgtagttta
     4801 taagtgtgta tattttaatt tataactttt ctaatatatg accaaaattt gttgatgtgc
     4861 aggttgttga taagggagct tctgctcagt ctttcatcga gagaatgacc aacttcgata
     4921 agaaccttcc aaacgagaag gttcttccaa agcactctct tctttacgag tacttcaccg
     4981 tttacaacga gcttaccaag gttaagtacg ttaccgaggg aatgagaaag ccagctttcc
     5041 tttctggaga gcagaagaag gctatcgttg atcttctttt caagaccaac agaaaggtta
     5101 ccgttaagca gttgaaggag gattacttca agaagatcga gtgcttcgat tctgttgaaa
     5161 tctctggagt tgaggataga ttcaacgctt ctcttggaac ctaccacgat cttttgaaga
     5221 tcatcaagga taaggatttc cttgataacg aggagaacga ggacatcctt gaggacatcg
     5281 ttcttaccct tacccttttc gaggatagag agatgatcga ggagagactc aagacctacg
     5341 ctcacctttt cgatgataag gttatgaagc agttgaagag aagaagatac accggatggg
     5401 gtagactttc tcgtaagttg atcaacggaa tcagagataa gcagtctgga aagaccatcc
     5461 ttgatttctt gaagtctgat ggattcgcta acagaaactt catgcagctt atccacgatg
     5521 attctcttac cttcaaggag gacatccaga aggctcaggt ttctggacag ggagattctc
     5581 ttcacgagca catcgctaac cttgctggat ctccagctat caagaaggga atccttcaga
     5641 ccgttaaggt tgttgatgag cttgttaagg ttatgggtag acacaagcca gagaacatcg
     5701 ttatcgagat ggctagagag aaccagacca cccagaaggg acagaagaac tctcgtgaga
     5761 gaatgaagag aatcgaggag ggaatcaagg agcttggatc tcaaatcttg aaggagcacc
     5821 cagttgagaa cacccagctt cagaacgaga agttgtacct ttactacctt cagaacggaa
     5881 gagatatgta cgttgatcag gagcttgaca tcaacagact ttctgattac gatgttgatc
     5941 acatcgttcc acagtctttc ttgaaggatg attctatcga taacaaggtt cttacccgtt
     6001 ctgataagaa cagaggaaag tctgataacg ttccatctga ggaggttgtt aagaagatga
     6061 agaactactg gagacagctt cttaacgcta agttgatcac ccagagaaag ttcgataacc
     6121 ttaccaaggc tgagagagga ggactttctg agcttgataa ggctggattc atcaagagac
     6181 agcttgttga gaccagacag atcaccaagc acgttgctca gatccttgat tctcgtatga
     6241 acaccaagta cgatgagaac gataagttga tcagagaggt taaggttatc accttgaagt
     6301 ctaagttggt ttctgatttc agaaaggatt tccagttcta caaggttaga gagatcaaca
     6361 actaccacca cgctcacgat gcttacctta acgctgttgt tggaaccgct cttatcaaga
     6421 agtacccaaa gttggagtct gagttcgttt acggagatta caaggtttac gatgttagaa
     6481 agatgatcgc taagtctgag caggagatcg gaaaggctac cgctaagtac ttcttctact
     6541 ctaacatcat gaacttcttc aagaccgaga tcacccttgc taacggagag atcagaaaga
     6601 gaccacttat cgagaccaac ggagagaccg gagagatcgt ttgggataag ggaagagatt
     6661 tcgctaccgt tagaaaggtt ctttctatgc cacaggttaa catcgttaag aaaaccgagg
     6721 ttcagaccgg aggattctct aaggagtcta tccttccaaa gagaaactct gataagttga
     6781 tcgctagaaa gaaggattgg gacccaaaga agtacggagg attcgattct ccaaccgttg
     6841 cttactctgt tcttgttgtt gctaaggttg agaagggaaa gtctaagaag ttgaagtctg
     6901 ttaaggagct tcttggaatc accatcatgg agcgttcttc tttcgagaag aacccaatcg
     6961 atttccttga ggctaaggga tacaaggagg ttaagaagga tcttatcatc aagttgccaa
     7021 agtactctct tttcgagctt gagaacggaa gaaagagaat gcttgcttct gctggagagc
     7081 ttcagaaggg aaacgagctt gctcttccat ctaagtacgt taacttcctt taccttgctt
     7141 ctcactacga gaagttgaag ggatctccag aggataacga gcagaagcag cttttcgttg
     7201 agcagcacaa gcactacctt gatgagatca tcgagcaaat ctctgagttc tctaagagag
     7261 ttatccttgc tgatgctaac cttgataagg ttctttctgc ttacaacaag cacagagata
     7321 agccaatcag agagcaggct gagaacatca tccacctttt cacccttacc aaccttggtg
     7381 ctccagctgc tttcaagtac ttcgatacca ccatcgatag aaaaagatac acctctacca
     7441 aggaggttct tgatgctacc cttatccacc agtctatcac cggactttac gagaccagaa
     7501 tcgatctttc tcagcttgga ggagataaga gaccagctgc taccaagaag gctggacagg
     7561 ctaagaagaa gaagtgaga
//
