LOCUS       sequence-181852-        3541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Module B, Promoter: 35S, Gene: SapI ccdb cassette for cloning 
            multiple gRNA spacers with Csy4 spacers , Terminator: 35S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3541)
  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 3541)
  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_B2101
FEATURES             Location/Qualifiers
     source          1..3541
                     /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"
     promoter        1068..1413
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     1356..1376
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     protein_bind    1430..1449
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     misc_RNA        1450..1525
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1526..1545
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     CDS             1865..2170
                     /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     2049..2068
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     polyA_signal    2198..2372
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        2449..2553
                     /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 agagcatgga gtcaaagatt
      901 caaatagagg acctaacaga actcgccgta aagactggcg aacagttcat acagagtctc
      961 ttacgactca atgacaagaa gaaaatcttc gtcaacatgg tggagcacga cacacttgtc
     1021 tactccaaaa atatcaaaga tacagtctca gaagaccaaa gggcaattga gacttttcaa
     1081 caaagggtaa tatccggaaa cctcctcgga ttccattgcc cagctatctg tcactttatt
     1141 gtgaagatag tggaaaagga aggtggctcc tacaaatgcc atcattgcga taaaggaaag
     1201 gccatcgttg aagatgcctc tgccgacagt ggtcccaaag atggaccccc acccacgagg
     1261 agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat
     1321 atctccactg acgtaaggga tgacgcacaa tcccactatc cttcgcaaga cccttcctct
     1381 atataaggaa gttcatttca tttggagaga acacggggga ctcctgcagg ttcactgccg
     1441 tataggcagg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     1501 tgaaaaagtg gcaccgagtc ggtgcgttca ctgccgtata ggcagaagcc agataacagt
     1561 atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa
     1621 gtatgtcaaa aggaggtatg ctatgaagca gcgtattaca gtgacagttg acagcgacag
     1681 ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg
     1741 cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg
     1801 gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa caggggctgg
     1861 tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
     1921 tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc
     1981 acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga
     2041 aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtttccgtta tcggggaaga
     2101 agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg
     2161 gggaatataa gctcttcagt cgatcgacaa gctcgagttt ctccataata atgtgtgagt
     2221 agttcccaga taagggaatt agggttccta tagggtttcg ctcatgtgtt gagcatataa
     2281 gaaaccctta gtatgtattt gtatttgtaa aatacttcta tcaataaaat ttctaattcc
     2341 taaaaccaaa atccagtact aaaatccaga tcccccgaat tagagctcta ccggtgacgc
     2401 aggtgatgaa aagccttagg gtcaggtggc acttttcggg gaaatgtgcg atgaacccct
     2461 atttgtttat ttttctaaat acattcaaat atgtatccgc taatgagaca ataaccctga
     2521 taaatgcttc aattatattg aaaaaggaag agtatgagta tccaacattt ccgtgtcgcc
     2581 cttattccct tttttgcggc cttctgcctt cctgtttttg ctcacccaga aacgctggtg
     2641 aaagtaaaag atgctgaaga tcagttgggt gcccgagtgg gttacattga actggatctc
     2701 aacagcggca aaatcttaga gagttttcgc cccgaagaac gcttcccaat gatgagcact
     2761 ttcaaagttc tgctatgtgg cgcggtgtta tcccgtattg atgctgggca agagcaactc
     2821 ggtcgccgca tacactattc tcagaatgac ttggttgagt attcaccagt cacagaaaag
     2881 catcttacgg atggcatgac agtaagagaa ttatgtagtg ctgccataac catgagtgat
     2941 aacactgcgg ccaacttact tctgacaacg attggaggac cgaaggagct aaccgctttt
     3001 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3061 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aactctccgc
     3121 aaactattaa ctggcgaact acttactctt gcttcacgcc agcaactcat tgactggatg
     3181 gaggcggaca aagttgcagg accacttctg cgctcggcac ttccggctgg ctggtttatt
     3241 gctgataaat ctggagccgg tgagcgtggc tctcgcggta tcattgcagc actggggcca
     3301 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3361 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3421 gacgaaggga aataaatagt agcccgcctg atgtgcgggc ttttttttgg tggtggttac
     3481 cagtgtcgct tagctaccgt aggtcctctt aggcggtccg attagcgaca tagtcatcaa
     3541 t
//
