LOCUS       sequence-181875-        4132 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Module B, Promoter: YAO, Gene: SapI ccdb cassette for cloning 
            multiple gRNA spacers with tRNA spacers , Terminator: 35S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4132)
  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 4132)
  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 Cell.
            2017 May 18. pii: tpc.00922.2016. doi: 10.1105/tpc.16.00922."
COMMENT     Sequence Label: pMOD_B2309
FEATURES             Location/Qualifiers
     source          1..4132
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     misc_RNA        1984..2059
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             2456..2761
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2640..2659
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     polyA_signal    2789..2963
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        3040..3144
                     /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 aagcgggaga gcgcccacct gccaggggac ggcgcgccga agagcgatgg gaaattcatt
      901 gaaaacccta aacccaaatc aacagctgca attcaaaagg ggactaattg acaaacaaaa
      961 attgataaca aatagaggta gggggagagt ttcgtacgcg acaatgagat tgagctcttg
     1021 aggacttgtg aagttgccaa cgcacgagtg agtgacactg gtcggtttgt gagccgtaac
     1081 aacgtagttc catgagctca tcttcctctt ctttgtctcc agggaatttg agttcgactt
     1141 tctacgcgag ggccctcgag gaagcttcta gatttctgaa tcgagctttc ggaattttaa
     1201 catagagaag ttagagagag aatgaaaagc caaaggaggc gaaaatcgaa caaggaagaa
     1261 gaaagacaac tttcgacaaa gactggtcgg tcggttttgg tagacaattg aaattagatg
     1321 gatggtccgg ttcggtatac tataagatta aaaacagttt taaattcagc taaaccgaac
     1381 tcatttgatt ttattaaacc ggaatcatcc gattcgagtt tgtaaaaaat accgaaattg
     1441 aaaacactaa acaaaaactg tattaaactg ttactgaaat aagagaatct cccaattcgg
     1501 tttacgtact actcttcaga aatcagaacc aaaaattcag aaatcggatt gaaccaaact
     1561 taaattgacg gtccggttag tcttcggctc tacaaattaa aggcccaagt ttctgcttta
     1621 aaagaacgaa atagttaatg ggctcaaacc atagaccagg taagtcatgg gcttggttag
     1681 tccgggtcaa cccggtagac ccgattcctg aagaaaacct agtggaaggt ttaaagttgt
     1741 aaactttccg accaaataaa caaaatcgtt ttccagcttc ttccgtcgcc actaaaccct
     1801 gaggctaaac ctagacgagt caaagtgtaa aatcgttaaa ccctaagagg gagtgagaga
     1861 gagaagaatg aagtacaaca acgagaagaa gaaaggagcc tgcaggaaca aagcaccagt
     1921 ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt
     1981 gcagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     2041 agtggcaccg agtcggtgca acaaagcacc agtggtctag tggtagaata gtaccctgcc
     2101 acggtacaga cccgggttcg attcccggct ggtgcaaagc cagataacag tatgcgtatt
     2161 tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa
     2221 aaggaggtat gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt
     2281 gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat gcagaatgaa
     2341 gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat ggctgaggtc
     2401 gcccggttta ttgaaatgaa cggctctttt gctgacgaga acaggggctg gtgaaatgca
     2461 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
     2521 tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
     2581 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
     2641 catgatgacc accgatatgg ccagtgtgcc ggtttccgtt atcggggaag aagtggctga
     2701 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
     2761 agctcttcag tcgatcgaca agctcgagtt tctccataat aatgtgtgag tagttcccag
     2821 ataagggaat tagggttcct atagggtttc gctcatgtgt tgagcatata agaaaccctt
     2881 agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc ctaaaaccaa
     2941 aatccagtac taaaatccag atcccccgaa ttagagctct accggtgacg caggtgatga
     3001 aaagccttag ggtcaggtgg cacttttcgg ggaaatgtgc gatgaacccc tatttgttta
     3061 tttttctaaa tacattcaaa tatgtatccg ctaatgagac aataaccctg ataaatgctt
     3121 caattatatt gaaaaaggaa gagtatgagt atccaacatt tccgtgtcgc ccttattccc
     3181 ttttttgcgg ccttctgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     3241 gatgctgaag atcagttggg tgcccgagtg ggttacattg aactggatct caacagcggc
     3301 aaaatcttag agagttttcg ccccgaagaa cgcttcccaa tgatgagcac tttcaaagtt
     3361 ctgctatgtg gcgcggtgtt atcccgtatt gatgctgggc aagagcaact cggtcgccgc
     3421 atacactatt ctcagaatga cttggttgag tattcaccag tcacagaaaa gcatcttacg
     3481 gatggcatga cagtaagaga attatgtagt gctgccataa ccatgagtga taacactgcg
     3541 gccaacttac ttctgacaac gattggagga ccgaaggagc taaccgcttt tttgcacaac
     3601 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3661 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caactctccg caaactatta
     3721 actggcgaac tacttactct tgcttcacgc cagcaactca ttgactggat ggaggcggac
     3781 aaagttgcag gaccacttct gcgctcggca cttccggctg gctggtttat tgctgataaa
     3841 tctggagccg gtgagcgtgg ctctcgcggt atcattgcag cactggggcc agatggtaag
     3901 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3961 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agacgaaggg
     4021 aaataaatag tagcccgcct gatgtgcggg cttttttttg gtggtggtta ccagtgtcgc
     4081 ttagctaccg taggtcctct taggcggtcc gattagcgac atagtcatca at
//
