LOCUS       Exported                9093 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Module A, Promoter: ZmUbi, Gene: Csy4-P2A-TaCas9_D10A nickase, 
            Terminator: HSP.
ACCESSION   .
VERSION     .
KEYWORDS    pMOD_A1610
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9093)
  AUTHORS   Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, 
            Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF
  TITLE     A multi-purpose toolkit to enable advanced genome engineering in 
            plants.
  JOURNAL   Plant Cell. 2017 May 18. pii: tpc.00922.2016. doi: 
            10.1105/tpc.16.00922.
  PUBMED    28522548
REFERENCE   2  (bases 1 to 9093)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9093
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      80..668
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     569..588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     822..839
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             3453..3509
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3510..7613
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7614..7661
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     promoter        8014..8105
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gccttgacgg caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
       61 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
      121 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ctaactcttt
      181 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
      241 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      301 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      361 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgctcacagc
      421 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      481 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      541 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      601 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      661 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      721 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
      781 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
      841 aagcggaaga gcgcccacct gctcatgatc tgcaggcgcg cctgcagtgc agcgtgaccc
      901 ggtcgtgccc ctctctagag ataatgagca ttgcatgtct aagttataaa aaattaccac
      961 atattttttt tgtcacactt gtttgaagtg cagtttatct atctttatac atatatttaa
     1021 actttactct acgaataata taatctatag tactacaata atatcagtgt tttagagaat
     1081 catataaatg aacagttaga catggtctaa aggacaattg agtattttga caacaggact
     1141 ctacagtttt atctttttag tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc
     1201 tatataatac ttcatccatt ttattagtac atccatttag ggtttagggt taatggtttt
     1261 tatagactaa tttttttagt acatctattt tattctattt tagcctctaa attaagaaaa
     1321 ctaaaactct attttagttt ttttatttaa taatttagat ataaaataga ataaaataaa
     1381 gtgactaaaa attaaacaaa taccctttaa gaaattaaaa aaactaagga aacatttttc
     1441 ttgtttcgag tagataatgc cagcctgtta aacgccgtcg acgagtctaa cggacaccaa
     1501 ccagcgaacc agcagcgtcg cgtcgggcca agcgaagcag acggcacggc atctctgtcg
     1561 ctgcctctgg acccctctcg agagttccgc tccaccgttg gacttgctcc gctgtcggca
     1621 tccagaaatt gcgtggcgga gcggcagacg tgagccggca cggcaggcgg cctcctcctc
     1681 ctctcacggc accggcagct acgggggatt cctttcccac cgctccttcg ctttcccttc
     1741 ctcgcccgcc gtaataaata gacaccccct ccacaccctc tttccccaac ctcgtgttgt
     1801 tcggagcgca cacacacaca accagatctc ccccaaatcc acccgtcggc acctccgctt
     1861 caaggtacgc cgctcgtcct cccccccccc ctctctacct tctctagatc ggcgttccgg
     1921 tccatggtta gggcccggta gttctacttc tgttcatgtt tgtgttagat ccgtgtttgt
     1981 gttagatccg tgctgctagc gttcgtacac ggatgcgacc tgtacgtcag acacgttctg
     2041 attgctaact tgccagtgtt tctctttggg gaatcctggg atggctctag ccgttccgca
     2101 gacgggatcg atttcatgat tttttttgtt tcgttgcata gggtttggtt tgcccttttc
     2161 ctttatttca atatatgccg tgcacttgtt tgtcgggtca tcttttcatg cttttttttg
     2221 tcttggttgt gatgatgtgg tctggttggg cggtcgttct agatcggagt agaattaatt
     2281 ctgtttcaaa ctacctggtg gatttattaa ttttggatct gtatgtgtgt gccatacata
     2341 ttcatagtta cgaattgaag atgatggatg gaaatatcga tctaggatag gtatacatgt
     2401 tgatgcgggt tttactgatg catatacaga gatgcttttt gttcgcttgg ttgtgatgat
     2461 gtggtgtggt tgggcggtcg ttcattcgtt ctagatcgga gtagaatact gtttcaaact
     2521 acctggtgta tttattaatt ttggaactgt atgtgtgtgt catacatctt catagttacg
     2581 agtttaagat ggatggaaat atcgatctag gataggtata catgttgatg tgggttttac
     2641 tgatgcatat acatgatggc atatgcagca tctattcata tgctctaacc ttgagtacct
     2701 atctattata ataaacaagt atgttttata attattttga tcttgatata cttggatgat
     2761 ggcatatgca gcagctatat gtggattttt ttagccctgc cttcatacgc tatttatttg
     2821 cttggtactg tttcttttgt cgatgctcac cctgttgttt ggtgttactt cctgcaggaa
     2881 gcatggacca ctacctcgac atcaggctca ggccagaccc agagttccca ccagcccagc
     2941 tcatgtccgt cctcttcggc aagctccacc aggccctcgt ggcccagggc ggcgacagga
     3001 tcggcgtgtc cttcccagac ctcgacgagt ccaggtccag gctcggcgag aggctccgca
     3061 tccacgcctc cgccgacgac ctcagggccc tcctcgccag gccgtggctg gagggcctca
     3121 gggaccacct ccagttcggc gagccagccg tggtgccaca cccaacccca tacaggcaag
     3181 tgtccagggt gcaagccaag tccaacccag agaggctcag gaggaggctc atgaggaggc
     3241 acgacctctc cgaggaagag gccaggaagc gcatcccaga caccgtggcc agggccctcg
     3301 acctcccatt cgtgaccctc aggtcccagt ccaccggcca gcacttccgc ctcttcatca
     3361 ggcacggccc actccaggtg accgccgagg agggcggctt tacctgctac ggcctctcca
     3421 agggcggctt cgtgccgtgg ttcggctccg gcgccaccaa cttctccctc ctcaagcaag
     3481 ccggcgacgt ggaggagaac ccaggcccaa tggacaagaa gtactcgatc ggcctcgcca
     3541 tcgggacgaa ctcagttggc tgggccgtga tcaccgacga gtacaaggtg ccctctaaga
     3601 agttcaaggt cctggggaac accgaccgcc attccatcaa gaagaacctc atcggcgctc
     3661 tcctgttcga cagcggggag accgctgagg ctacgaggct caagagaacc gctaggcgcc
     3721 ggtacacgag aaggaagaac aggatctgct acctccaaga gattttctcc aacgagatgg
     3781 ccaaggttga cgattcattc ttccaccgcc tggaggagtc tttcctcgtg gaggaggata
     3841 agaagcacga gcggcatccc atcttcggca acatcgtgga cgaggttgcc taccacgaga
     3901 agtaccctac gatctaccat ctgcggaaga agctcgtgga ctccaccgat aaggcggacc
     3961 tcagactgat ctacctcgct ctggcccaca tgatcaagtt ccgcggccat ttcctgatcg
     4021 agggggatct caacccagac aacagcgatg ttgacaagct gttcatccaa ctcgtgcaga
     4081 cctacaacca actcttcgag gagaacccga tcaacgcctc tggcgtggac gcgaaggcta
     4141 tcctgtccgc gaggctctcg aagtccagga ggctggagaa cctgatcgct cagctcccag
     4201 gcgagaagaa gaacggcctg ttcgggaacc tcatcgctct cagcctgggg ctcaccccga
     4261 acttcaagtc gaacttcgat ctcgctgagg acgccaagct gcaactctcc aaggacacct
     4321 acgacgatga cctcgataac ctcctggccc agatcggcga tcaatacgcg gacctgttcc
     4381 tcgctgccaa gaacctgtcg gacgccatcc tcctgtcaga tatcctccgc gtgaacaccg
     4441 agatcacgaa ggctccactc tctgcctcca tgatcaagcg ctacgacgag caccatcagg
     4501 atctgaccct cctgaaggcg ctggtccgcc aacagctccc ggagaagtac aaggagattt
     4561 tcttcgatca gtcgaagaac ggctacgctg ggtacatcga cggcggggcc tcacaagagg
     4621 agttctacaa gttcatcaag ccaatcctgg agaagatgga cggcacggag gagctcctgg
     4681 tgaagctcaa cagggaggac ctcctgcgga agcagagaac cttcgataac ggcagcatcc
     4741 cccaccaaat ccatctcggg gagctgcacg ccatcctgag aaggcaagag gacttctacc
     4801 ctttcctcaa ggataaccgg gagaagatcg agaagatcct gaccttcaga atcccatact
     4861 acgtcggccc tctcgcgcgg gggaactcaa gattcgcttg gatgacccgc aagtctgagg
     4921 agaccatcac gccgtggaac ttcgaggagg tggtggacaa gggcgctagc gctcagtcgt
     4981 tcatcgagag gatgaccaac ttcgacaaga acctgcccaa cgagaaggtg ctccctaagc
     5041 actcgctcct gtacgagtac ttcaccgtct acaacgagct cacgaaggtg aagtacgtca
     5101 ccgagggcat gcgcaagcca gcgttcctgt ccggggagca gaagaaggct atcgtggacc
     5161 tcctgttcaa gaccaaccgg aaggtcacgg ttaagcaact caaggaggac tacttcaaga
     5221 agatcgagtg cttcgattcg gtcgagatca gcggcgttga ggaccgcttc aacgccagcc
     5281 tcgggaccta ccacgatctc ctgaagatca tcaaggataa ggacttcctg gacaacgagg
     5341 agaacgagga tatcctggag gacatcgtgc tgaccctcac gctgttcgag gacagggaga
     5401 tgatcgagga gcgcctgaag acgtacgccc atctcttcga tgacaaggtc atgaagcaac
     5461 tcaagcgccg gagatacacc ggctggggga ggctgtcccg caagctcatc aacggcatcc
     5521 gggacaagca gtccgggaag accatcctcg acttcctcaa gagcgatggc ttcgccaaca
     5581 ggaacttcat gcaactgatc cacgatgaca gcctcacctt caaggaggat atccaaaagg
     5641 ctcaagtgag cggccagggg gactcgctgc acgagcatat cgcgaacctc gctggctccc
     5701 ccgcgatcaa gaagggcatc ctccagaccg tgaaggttgt ggacgagctc gtgaaggtca
     5761 tgggccggca caagcctgag aacatcgtca tcgagatggc cagagagaac caaaccacgc
     5821 agaaggggca aaagaactct agggagcgca tgaagcgcat cgaggagggc atcaaggagc
     5881 tggggtccca aatcctcaag gagcacccag tggagaacac ccaactgcag aacgagaagc
     5941 tctacctgta ctacctccag aacggcaggg atatgtacgt ggaccaagag ctggatatca
     6001 accgcctcag cgattacgac gtcgatcata tcgttcccca gtctttcctg aaggatgact
     6061 ccatcgacaa caaggtcctc accaggtcgg acaagaaccg cggcaagtca gataacgttc
     6121 catctgagga ggtcgttaag aagatgaaga actactggag gcagctcctg aacgccaagc
     6181 tgatcacgca aaggaagttc gacaacctca ccaaggctga gagaggcggg ctctcagagc
     6241 tggacaaggc cggcttcatc aagcggcagc tggtcgagac cagacaaatc acgaagcacg
     6301 ttgcgcaaat cctcgactct cggatgaaca cgaagtacga tgagaacgac aagctgatca
     6361 gggaggttaa ggtgatcacc ctgaagtcta agctcgtctc cgacttcagg aaggatttcc
     6421 agttctacaa ggttcgcgag atcaacaact accaccatgc ccatgacgct tacctcaacg
     6481 ctgtggtcgg caccgctctg atcaagaagt acccaaagct ggagtccgag ttcgtgtacg
     6541 gggactacaa ggtttacgat gtgcgcaaga tgatcgccaa gtcggagcaa gagatcggca
     6601 aggctaccgc caagtacttc ttctactcaa acatcatgaa cttcttcaag accgagatca
     6661 cgctggccaa cggcgagatc cggaagagac cgctcatcga gaccaacggc gagacggggg
     6721 agatcgtgtg ggacaagggc agggatttcg cgaccgtccg caaggttctc tccatgcccc
     6781 aggtgaacat cgtcaagaag accgaggtcc aaacgggcgg gttctcaaag gagtctatcc
     6841 tgcctaagcg gaacagcgac aagctcatcg ccagaaagaa ggactgggac ccaaagaagt
     6901 acggcgggtt cgacagccct accgtggcct actcggtcct ggttgtggcg aaggttgaga
     6961 agggcaagtc caagaagctc aagagcgtga aggagctcct ggggatcacc atcatggaga
     7021 ggtccagctt cgagaagaac ccaatcgact tcctggaggc caagggctac aaggaggtga
     7081 agaaggacct gatcatcaag ctcccgaagt actctctctt cgagctggag aacggcagga
     7141 agagaatgct ggcttccgct ggcgagctcc agaaggggaa cgagctcgcg ctgccaagca
     7201 agtacgtgaa cttcctctac ctggcttccc actacgagaa gctcaagggc agcccggagg
     7261 acaacgagca aaagcagctg ttcgtcgagc agcacaagca ttacctcgac gagatcatcg
     7321 agcaaatctc cgagttcagc aagcgcgtga tcctcgccga cgcgaacctg gataaggtcc
     7381 tctccgccta caacaagcac cgggacaagc ccatcagaga gcaagcggag aacatcatcc
     7441 atctcttcac cctgacgaac ctcggcgctc ctgctgcttt caagtacttc gacaccacga
     7501 tcgatcggaa gagatacacc tccacgaagg aggtcctgga cgcgaccctc atccaccagt
     7561 cgatcaccgg cctgtacgag acgaggatcg acctctcaca actcggcggg gataagagac
     7621 ccgcagcaac caagaaggca gggcaagcaa agaagaagaa gtgactcgag tatatgaaga
     7681 tgaagatgaa atatttggtg tgtcaaataa aaagcttgtg tgcttaagtt tgtgtttttt
     7741 tcttggcttg ttgtgttatg aatttgtggc tttttctaat attaaatgaa tgtaagatca
     7801 cattataatg aataaacaaa tgtttctata atccattgtg aatgttttgt tggatctctt
     7861 ctgcagcata taactactgt atgtgctatg gtatggacta tggaatatga ttaaagataa
     7921 ggagctcgtt aaccggtgac ggacgtgggc aggtgtgatg aaaagcctta gggtcaggtg
     7981 gcacttttcg gggaaatgtg cgatgaaccc ctatttgttt atttttctaa atacattcaa
     8041 atatgtatcc gctaatgaga caataaccct gataaatgct tcaattatat tgaaaaagga
     8101 agagtatgag tatccaacat ttccgtgtcg cccttattcc cttttttgcg gccttctgcc
     8161 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     8221 gtgcccgagt gggttacatt gaactggatc tcaacagcgg caaaatctta gagagttttc
     8281 gccccgaaga acgcttccca atgatgagca ctttcaaagt tctgctatgt ggcgcggtgt
     8341 tatcccgtat tgatgctggg caagagcaac tcggtcgccg catacactat tctcagaatg
     8401 acttggttga gtattcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     8461 aattatgtag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     8521 cgattggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     8581 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     8641 cgatgcctgt agcaatggca acaactctcc gcaaactatt aactggcgaa ctacttactc
     8701 ttgcttcacg ccagcaactc attgactgga tggaggcgga caaagttgca ggaccacttc
     8761 tgcgctcggc acttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     8821 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     8881 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     8941 gtgcctcact gattaagcat tggtaactgt cagacgaagg gaaataaata gtagcccgcc
     9001 tgatgtgcgg gctttttttt ggtggtggtt accagtgtcg cttagctacc gtaggtcctc
     9061 ttaggcggtc cgattagcga catagtcatc aat
//
