LOCUS       sequence-372989-        3656 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 3656)
  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 3656)
  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-DT3T4
FEATURES             Location/Qualifiers
     source          1..3656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     235..254
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(669..971)
                     /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     complement(771..790)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     1094..1116
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1108..1125
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1109..1125
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1132..1151
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1132..1150
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        complement(1808..1883)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1954..1972)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1955..1972)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1990..2006)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1990..2006)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2003..2025)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        complement(2045..2092)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(2103..2126)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(2134..2161)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2253..2339)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(2504..3092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2584..2603)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3317..3656,1..320))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca
       61 ctatcccata tcaccagctc accgtctttc attgccatac ggaactccgg atgagcattc
      121 atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttact tttctttacg
      181 gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact
      241 gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat
      301 ccagtgattt ttttctccat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta
      361 acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg
      421 gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc
      481 actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata
      541 cgctacttgc attacagctt acgaaccgaa cgaggcttat gtccactcat ggtccgtttc
      601 tgcggactgg ctttctacgt gaaaaggatc taggtgaaga tcctttttga taatctcatg
      661 cctgacattt atattcccca gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg
      721 tggctgagat cagccacttc ttccccgata acggatactg gcacacttgc catatcggtg
      781 gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc acgggagact
      841 ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc cggcgtgtca
      901 ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt ataggtgtaa
      961 accttaaact gccgtacgta taggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     1021 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     1081 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac
     1141 tcactatagg gcgaattggg ccaggccaaa taggccaaag gattattggt ctctaaacca
     1201 tcttccatat ctcttctcaa tcactacttc gactctagct gtatataaac tcagcttcgt
     1261 tttcttatct aagcgatgtg ggacttttga agattgtttt caacttaaat gggcctatat
     1321 aagaaatact attgttcttt cccatataaa tgggcctgct tctcttcttt cagattccca
     1381 ggggcctttt gaagattatc ttcatatctt aagaatgaag atgttttatt caatcaaatt
     1441 cttgaaggtt cgatgcctaa tcattctaat cctgggacaa actatgaaac aagatacaaa
     1501 aactccgaat ggaaagttaa aaagaagaaa acgaaagcta cggttcaaga aaatgtaagc
     1561 tgataaacaa aaaaaaactg tatgaacgaa gaagaagaaa aaaagctaag aagaaatgat
     1621 gtattgtgcg gaaggcaagt cgaacggacc aatcactttg tcttagctat ctaaataagc
     1681 tcattgcagg tccaactttt ttcgcgcaga tttgcatcca aaacttcact tatgattcac
     1741 gaaacggaaa aaaaaaccca gaaatctaca gatgaagaaa aaatctgaaa atttttgcca
     1801 aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa
     1861 cttgctattt ctagctctaa aacgatctag atttcccggt aacaatagag accattattc
     1921 cttggcctta agggccttgc atagcttgag tattctatag tgtcacctaa atagcttggc
     1981 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcggtt tagttcctca
     2041 ccttgtcgta ttatactatg ccgatatact atgccgatga ttaattgtca acacttaagt
     2101 aactaactaa caggaagagt ttgtagaaac gcaaaaaggc catccgtcag gatggccttc
     2161 tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt
     2221 tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc
     2281 gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg ttttatttga
     2341 tgcctggcag ttccctactc tcgcttagta gttagacgtg cccgagatcc atcctagcgg
     2401 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2461 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2521 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2581 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2641 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2701 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2761 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2821 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2881 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2941 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3001 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3061 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3121 ctgacgctca gtggaacggg gcgggcggcg ttgtgacaat ttaccgaaca actccgcggg
     3181 acttgacctg atagtttggc tgtgagcaat tatgtgctta gtgcatctaa cgcttgagtt
     3241 aagccgcgcc gcgaagcggc gtcggcttga acgaatttct agctagagga tcgcaccaat
     3301 aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat
     3361 taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
     3421 gcatcagcac cttgtcgcct tgcgtataat atttgcccat tgtgaaaacg ggggcgaaga
     3481 agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg
     3541 agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac
     3601 acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattca
//
