LOCUS       sequence-181864-        3245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Module B, Promoter: CmYLCV, Gene: SapI ccdb cassette for cloning 
            multiple gRNA spacers with Csy4 spacers (only works with 
            pMOD_C2200), Terminator: none - to be fused to gRNA array in MODULE 
            C .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3245)
  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 3245)
  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_B2203
FEATURES             Location/Qualifiers
     source          1..3245
                     /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"
     protein_bind    1358..1377
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        1378..1453
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1454..1473
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     CDS             1793..2098
                     /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     1977..1996
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     promoter        2153..2257
                     /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 agagctggca gacatactgt
      901 cccacaaatg aagatggaat ctgtaaaaga aaacgcgtga aataatgcgt ctgacaaagg
      961 ttaggtcggc tgcctttaat caataccaaa gtggtcccta ccacgatgga aaaactgtgc
     1021 agtcggtttg gctttttctg acgaacaaat aagattcgtg gccgacaggt gggggtccac
     1081 catgtgaagg catcttcaga ctccaataat ggagcaatga cgtaagggct tacgaaataa
     1141 gtaagggtag tttgggaaat gtccactcac ccgtcagtct ataaatactt agcccctccc
     1201 tcattgttaa gggagcaaaa tctcagagag atagtcctag agagagaaag agagcaagta
     1261 gcctagaagt agtcaaggcg gcgaagtatt caggcacgtg gccaggaaga agaaaagcca
     1321 agacgacgaa aacaggtaag agctaagctt cctgcaggtt cactgccgta taggcaggtt
     1381 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     1441 accgagtcgg tgcgttcact gccgtatagg cagaagccag ataacagtat gcgtatttgc
     1501 gcgctgattt ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaag
     1561 gaggtatgct atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct
     1621 caaggcatat atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc
     1681 cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc
     1741 cggtttattg aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt
     1801 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga
     1861 tattattgac acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc
     1921 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat
     1981 gatgaccacc gatatggcca gtgtgccggt ttccgttatc ggggaagaag tggctgatct
     2041 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataagc
     2101 tcttcagtcc gcaggtgcct tagggtcagg tggcactttt cggggaaatg tgcgatgaac
     2161 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctaatga gacaataacc
     2221 ctgataaatg cttcaattat attgaaaaag gaagagtatg agtatccaac atttccgtgt
     2281 cgcccttatt cccttttttg cggccttctg ccttcctgtt tttgctcacc cagaaacgct
     2341 ggtgaaagta aaagatgctg aagatcagtt gggtgcccga gtgggttaca ttgaactgga
     2401 tctcaacagc ggcaaaatct tagagagttt tcgccccgaa gaacgcttcc caatgatgag
     2461 cactttcaaa gttctgctat gtggcgcggt gttatcccgt attgatgctg ggcaagagca
     2521 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtattcac cagtcacaga
     2581 aaagcatctt acggatggca tgacagtaag agaattatgt agtgctgcca taaccatgag
     2641 tgataacact gcggccaact tacttctgac aacgattgga ggaccgaagg agctaaccgc
     2701 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     2761 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaactct
     2821 ccgcaaacta ttaactggcg aactacttac tcttgcttca cgccagcaac tcattgactg
     2881 gatggaggcg gacaaagttg caggaccact tctgcgctcg gcacttccgg ctggctggtt
     2941 tattgctgat aaatctggag ccggtgagcg tggctctcgc ggtatcattg cagcactggg
     3001 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3061 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3121 gtcagacgaa gggaaataaa tagtagcccg cctgatgtgc gggctttttt ttggtggtgg
     3181 ttaccagtgt cgcttagcta ccgtaggtcc tcttaggcgg tccgattagc gacatagtca
     3241 tcaat
//
