LOCUS       sequence-75674-e        3762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas based plant genome editing and gene regulation; used as 
            template for making expression cassette with multiple gRNA target 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3762)
  AUTHORS   Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ
  TITLE     A CRISPR/Cas9 toolkit for multiplex genome editing in plants.
  JOURNAL   BMC Plant Biol. 2014 Nov 29;14(1):327.
  PUBMED    25432517
REFERENCE   2  (bases 1 to 3762)
  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: "BMC Plant 
            Biol."; date: "2014-11-29"; volume: "14(1)"; pages: "327"
COMMENT     Sequence Label: pCBC-MT2T3
FEATURES             Location/Qualifiers
     source          1..3762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      85..673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     574..593
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      838..924
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1016..1043
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     1051..1074
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     promoter        1085..1132
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     1152..1174
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1171..1187
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1171..1187
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1205..1223
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1205..1222
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        1278..1353
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2132..2151)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2133..2151)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2158..2175)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2158..2174)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2167..2189)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2312..2614
                     /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="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2493..2512
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2963..3622
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3029..3048)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 cggagttgtt cggtaaattg tcacaacgcc gcccgccccg ttccactgag cgtcagaccc
       61 ggtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
      121 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
      181 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
      241 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
      301 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
      361 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
      421 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
      481 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
      541 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
      601 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
      661 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
      721 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
      781 taggatggat ctcgggcacg tctaactact aagcgagagt agggaactgc caggcatcaa
      841 ataaaacgaa aggctcagtc ggaagactgg gcctttcgtt ttatctgttg tttgtcggtg
      901 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgt gaagcaacgg
      961 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaac taagcagaag
     1021 gccatcctga cggatggcct ttttgcgttt ctacaaactc ttcctgttag ttagttactt
     1081 aagtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     1141 gaactaaacc gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     1201 agctatttag gtgacactat agaatactca agctatgcaa ggcccttaag gccaaggggg
     1261 cttcgtgcca ccaacgagtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     1321 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt tttgtccttc tgttttttta
     1381 gtcagtctct tttttcagaa gtacaacatc ttttttttgt ccttctgttt ttttagtcag
     1441 tctctttttt cagaagtaca acatcttttt tttgtccttc tgttttttta gtcagtctct
     1501 tttttcagaa gtacaacatc ttttttttgt ccttctgttt ttttagtcag tctctttttt
     1561 cagaagtact ctatgatgta tcgttctggg aaatgtctgt ctgtctacaa ccccataatc
     1621 tatatttgca atcacacatc taatattctc tgtgacaaga cagccgaaca cgacttgcct
     1681 tccgcacaat acatcatttc ttcttagctt tttttcttct tcttcgttca tacagttttt
     1741 ttttgtttat cagcttacat tttcttgaac cgtagctttc gttttcttct ttttaacttt
     1801 ccattcggag tttttgtatc ttgtttcata gtttgtccca ggattagaat gattaggcat
     1861 cgaaccttca agaatttgat tgaataaaac atcttcattc ttaagatatg aagataatct
     1921 tcaaaaggcc cctgggaatc tgaaagaaga gaagcaggcc catttatatg ggaaagaaca
     1981 atagtatttc ttatataggc ccatttaagt tgaaaacaat cttcaaaagt cccacatcgc
     2041 ttagataaga aaacgaagct gagtttatat acagctagag tcgaagtagt gatatctaat
     2101 acctttggcc tatttggcct ggcccaattc gccctatagt gagtcgtatt acaattcact
     2161 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     2221 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     2281 ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga
     2341 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg
     2401 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta
     2461 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggcaagtgt
     2521 gccagtatcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
     2581 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag
     2641 gatcttcacc tagatccttt tcacgtagaa agccagtccg cagaaacgga ccatgagtgg
     2701 acataagcct cgttcggttc gtaagctgta atgcaagtag cgtatgcgct cacgcaactg
     2761 gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg
     2821 ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc aagcgcgtta
     2881 cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc agggcagtcg
     2941 ccctaaaaca aagttaaaca tcatggagaa aaaaatcact ggatatacca ccgttgatat
     3001 atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta
     3061 taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga aaagtaagca
     3121 caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggagtt
     3181 ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac
     3241 cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt
     3301 ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta
     3361 tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt
     3421 caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcacaat
     3481 gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca
     3541 tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga
     3601 tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgcgatc ctctagctag
     3661 aaattcgttc aagccgacgc cgcttcgcgg cgcggcttaa ctcaagcgtt agatgcacta
     3721 agcacataat tgctcacagc caaactatca ggtcaagtcc cg
//
