LOCUS       sequence-391100-        6390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Phytobrick (MoClo) Level 0 Part.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6390)
  AUTHORS   Chamness JC, Kumar J, Cruz AJ, Rhuby E, Holum MJ, Cody JP, Tibebu R,
            Gamo ME, Starker CG, Zhang F, Voytas DF
  TITLE     An extensible vector toolkit and parts library for advanced 
            engineering of plant genomes.
  JOURNAL   Plant Genome. 2023 Mar 9:e20312. doi: 10.1002/tpg2.20312.
  PUBMED    36896468
REFERENCE   2  (bases 1 to 6390)
  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 
            Genome. 2023 Mar 9:e20312. doi: 10.1002/tpg2.20312."
COMMENT     Sequence Label: pMC-6-AtSpCas9-9
FEATURES             Location/Qualifiers
     source          1..6390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             91..4194
                     /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"
     CDS             91..4194
                     /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"
     CDS             4207..4227
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4270..4288)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4356..4460
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4682..5473
                     /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      5644..6232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6133..6152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(6386..6390,1..13)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
       61 gccgattcat taatcactct gtggtctcaa atggataaga agtactctat cggactcgat
      121 atcggaacta actctgtggg atgggctgtg atcaccgatg agtacaaggt gccatctaag
      181 aagttcaagg ttctcggaaa caccgatagg cactctatca agaaaaacct tatcggtgct
      241 ctcctcttcg attctggtga aactgctgag gctaccagac tcaagagaac cgctagaaga
      301 aggtacacca gaagaaagaa caggatctgc tacctccaag agatcttctc taacgagatg
      361 gctaaagtgg atgattcatt cttccacagg ctcgaagagt cattcctcgt ggaagaagat
      421 aagaagcacg agaggcaccc tatcttcgga aacatcgttg atgaggtggc ataccacgag
      481 aagtacccta ctatctacca cctcagaaag aagctcgttg attctactga taaggctgat
      541 ctcaggctca tctacctcgc tctcgctcac atgatcaagt tcagaggaca cttcctcatc
      601 gagggtgatc tcaaccctga taactctgat gtggataagt tgttcatcca gctcgtgcag
      661 acctacaacc agcttttcga agagaaccct atcaacgctt caggtgtgga tgctaaggct
      721 atcctctctg ctaggctctc taagtcaaga aggcttgaga acctcattgc tcagctccct
      781 ggtgagaaga agaacggact tttcggaaac ttgatcgctc tctctctcgg actcacccct
      841 aacttcaagt ctaacttcga tctcgctgag gatgcaaagc tccagctctc aaaggatacc
      901 tacgatgatg atctcgataa cctcctcgct cagatcggag atcagtacgc tgatttgttc
      961 ctcgctgcta agaacctctc tgatgctatc ctcctcagtg atatcctcag agtgaacacc
     1021 gagatcacca aggctccact ctcagcttct atgatcaaga gatacgatga gcaccaccag
     1081 gatctcacac ttctcaaggc tcttgttaga cagcagctcc cagagaagta caaagagatt
     1141 ttcttcgatc agtctaagaa cggatacgct ggttacatcg atggtggtgc atctcaagaa
     1201 gagttctaca agttcatcaa gcctatcctc gagaagatgg atggaaccga ggaactcctc
     1261 gtgaagctca atagagagga tcttctcaga aagcagagga ccttcgataa cggatctatc
     1321 cctcatcaga tccacctcgg agagttgcac gctatcctta gaaggcaaga ggatttctac
     1381 ccattcctca aggataacag ggaaaagatt gagaagattc tcaccttcag aatcccttac
     1441 tacgtgggac ctctcgctag aggaaactca agattcgctt ggatgaccag aaagtctgag
     1501 gaaaccatca ccccttggaa cttcgaagag gtggtggata agggtgctag tgctcagtct
     1561 ttcatcgaga ggatgaccaa cttcgataag aaccttccaa acgagaaggt gctccctaag
     1621 cactctttgc tctacgagta cttcaccgtg tacaacgagt tgaccaaggt taagtacgtg
     1681 accgagggaa tgaggaagcc tgcttttttg tcaggtgagc aaaagaaggc tatcgttgat
     1741 ctcttgttca agaccaacag aaaggtgacc gtgaagcagc tcaaagagga ttacttcaag
     1801 aaaatcgagt gcttcgattc agttgagatt tctggtgttg aggataggtt caacgcatct
     1861 ctcggaacct accacgatct cctcaagatc attaaggata aggatttctt ggataacgag
     1921 gaaaacgagg atatcttgga ggatatcgtt cttaccctca ccctctttga agatagagag
     1981 atgattgaag aaaggctcaa gacctacgct catctcttcg atgataaggt gatgaagcag
     2041 ttgaagagaa gaagatacac tggttgggga aggctctcaa gaaagctcat taacggaatc
     2101 agggataagc agtctggaaa gacaatcctt gatttcctca agtctgatgg attcgctaac
     2161 agaaacttca tgcagctcat ccacgatgat tctctcacct ttaaagagga tatccagaag
     2221 gctcaggttt caggacaggg tgatagtctc catgagcata tcgctaacct cgctggatct
     2281 cctgcaatca agaagggaat cctccagact gtgaaggttg tggatgagtt ggtgaaggtg
     2341 atgggaaggc ataagcctga gaacatcgtg atcgaaatgg ctagagagaa ccagaccact
     2401 cagaagggac agaagaactc tagggaaagg atgaagagga tcgaggaagg tatcaaagag
     2461 cttggatctc agatcctcaa agagcaccct gttgagaaca ctcagctcca gaatgagaag
     2521 ctctacctct actacctcca gaacggaagg gatatgtatg tggatcaaga gttggatatc
     2581 aacaggctct ctgattacga tgttgatcat atcgtgccac agtcattctt gaaggatgat
     2641 tctatcgata acaaggtgct caccaggtct gataagaaca ggggtaagag tgataacgtg
     2701 ccaagtgaag aggttgtgaa gaaaatgaag aactattgga ggcagctcct caacgctaag
     2761 ctcatcactc agagaaagtt cgataacttg actaaggctg agaggggagg actctctgaa
     2821 ttggataagg caggattcat caagaggcag cttgtggaaa ccaggcagat cactaagcac
     2881 gttgcacaga tcctcgattc taggatgaac accaagtacg atgagaacga taagttgatc
     2941 agggaagtga aggttatcac cctcaagtca aagctcgtgt ctgatttcag aaaggatttc
     3001 caattctaca aggtgaggga aatcaacaac taccaccacg ctcacgatgc ttaccttaac
     3061 gctgttgttg gaaccgctct catcaagaag tatcctaagc tcgagtcaga gttcgtgtac
     3121 ggtgattaca aggtgtacga tgtgaggaag atgatcgcta agtctgagca agagatcgga
     3181 aaggctaccg ctaagtattt cttctactct aacatcatga atttcttcaa gaccgagatt
     3241 accctcgcta acggtgagat cagaaagagg ccactcatcg agacaaacgg tgaaacaggt
     3301 gagatcgtgt gggataaggg aagggatttc gctaccgtta gaaaggtgct ctctatgcca
     3361 caggtgaaca tcgttaagaa aaccgaggtg cagaccggtg gattctctaa agagtctatc
     3421 ctccctaaga ggaactctga taagctcatt gctaggaaga aggattggga ccctaagaaa
     3481 tacggtggtt tcgattctcc taccgtggct tactctgttc tcgttgtggc taaggttgag
     3541 aagggaaaga gtaagaagct caagtctgtt aaggaacttc tcggaatcac tatcatggaa
     3601 aggtcatctt tcgagaagaa cccaatcgat ttcctcgagg ctaagggata caaagaggtt
     3661 aagaaggatc tcatcatcaa gctcccaaag tactcactct tcgaactcga gaacggtaga
     3721 aagaggatgc tcgcttctgc tggtgagctt caaaagggaa acgagcttgc tctcccatct
     3781 aagtacgtta actttcttta cctcgcttct cactacgaga agttgaaggg atctccagaa
     3841 gataacgagc agaagcaact tttcgttgag cagcacaagc actacttgga tgagatcatc
     3901 gagcagatct ctgagttctc taaaagggtg atcctcgctg atgcaaacct cgataaggtg
     3961 ttgtctgctt acaacaagca cagagataag cctatcaggg aacaggcaga gaacatcatc
     4021 catctcttca cccttaccaa cctcggtgct cctgctgctt tcaagtactt cgatacaacc
     4081 atcgatagga agagatacac ctctaccaaa gaagtgctcg atgctaccct catccatcag
     4141 tctatcactg gactctacga gactaggatc gatctctcac agctcggtgg tgattcaagg
     4201 gctgatccta agaagaagag gaaggtttga gctttgagac cacgaagtgg ctcttcagtg
     4261 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4321 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4381 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4441 aatattgaaa aaggaagagt atgcgctcac gcaactggtc cagaaccttg accgaacgca
     4501 gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttttggggt
     4561 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     4621 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     4681 catgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     4741 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     4801 cggcctgaag ccacacagcg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     4861 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     4921 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     4981 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     5041 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     5101 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     5161 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     5221 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     5281 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     5341 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     5401 ctcgcgcgca gatcagttgg aagaatttgt ccattacgta aaaggcgaga tcaccaaggt
     5461 agtcggcaaa taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     5521 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5581 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5641 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5701 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5761 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5821 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5881 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5941 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6001 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6061 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     6301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     6361 tcgccgcagc cgaacgaccg agcgcagcga
//
