LOCUS       sequence-451910-        3849 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 3849)
  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 3849)
  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-MT1T2
FEATURES             Location/Qualifiers
     source          1..3849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             158..817
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(224..243)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      1042..1630
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1531..1550
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      1795..1881
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1973..2000
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     2008..2031
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     promoter        2042..2089
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     2109..2131
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2128..2144
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2128..2144
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2162..2180
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2162..2179
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        2235..2310
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(3176..3195)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3177..3195)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3202..3219)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3202..3218)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3211..3233)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             3356..3658
                     /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     3537..3556
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
ORIGIN
        1 ggttttcatg gcttgttatg actgtttttt tggggtacag tctatgcctc gggcatccaa
       61 gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac
      121 gcagcagggc agtcgcccta aaacaaagtt aaacatcatg gagaaaaaaa tcactggata
      181 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
      241 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
      301 aaagaaaagt aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
      361 tgctcatccg gagttccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
      421 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
      481 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
      541 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
      601 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
      661 ccccgttttc acaatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
      721 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
      781 acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg
      841 cgatcctcta gctagaaatt cgttcaagcc gacgccgctt cgcggcgcgg cttaactcaa
      901 gcgttagatg cactaagcac ataattgctc acagccaaac tatcaggtca agtcccgcgg
      961 agttgttcgg taaattgtca caacgccgcc cgccccgttc cactgagcgt cagaccccgt
     1021 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1081 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1141 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     1201 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1261 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1321 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1381 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     1441 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     1501 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     1561 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1621 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1681 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgctag
     1741 gatggatctc gggcacgtct aactactaag cgagagtagg gaactgccag gcatcaaata
     1801 aaacgaaagg ctcagtcgga agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     1861 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgtgaa gcaacggccc
     1921 ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaactaa gcagaaggcc
     1981 atcctgacgg atggcctttt tgcgtttcta caaactcttc ctgttagtta gttacttaag
     2041 tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa
     2101 ctaaaccgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     2161 tatttaggtg acactataga atactcaagc tatgcaaggc ccttaaggcc aaggggcttc
     2221 gtgccaacca acgagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     2281 caacttgaaa aagtggcacc gagtcggtgc tttttttttt cgttttgcat tgagttttct
     2341 ccgtcgcatg tttgcagttt tattttccgt tttgcattga aatttctccg tctcatgttt
     2401 gcagcgtgtt caaaaagtac gcagctgtat ttcacttatt tacggcgcca cattttcatg
     2461 ccgtttgtgc caactatccc gagctagtga atacagcttg gcttcacaca acactggtga
     2521 cccgctgacc tgctcgtacc tcgtaccgtc gtacggcaca gcatttggaa ttaaagggtg
     2581 tgatcgatac tgcttgctgc tcatgaatcc aaaccacacg gagttcaaat tcccacagat
     2641 taaggctcgt ccgtcgcaca aggtaatgtg tgaatattat atctgtcgtg caaaattgcc
     2701 tggcctgcac aattgctgtt atagttggcg gcagggagag ttttaacatt gactagcgtg
     2761 ctgataattt gtgagaaata ataattgaca agtagatact gacatttgag aagagcttct
     2821 gaactgttat tagtaacaaa aatggaaagc tgatgcacgg aaaaaggaaa gaaaaagcca
     2881 tacttttttt taggtaggaa aagaaaaagc catacgagac tgatgtctct cagatgggcc
     2941 gggatctgtc tatctagcag gcagcagccc accaacctca cgggccagca attacgagtc
     3001 cttctaaaag ctcccgccga ggggcgctgg cgctgctgtg cagcagcacg tctaacatta
     3061 gtcccacctc gccagtttac agggagcaga accagcttat aagcggaggc gcggcaccaa
     3121 gaagcgtgac caggaggagc atgccccttt ggcctatttg gcctggccca attcgcccta
     3181 tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3241 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3301 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt
     3361 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga
     3421 tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc
     3481 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat
     3541 gatgaccacc gatatggcaa gtgtgccagt atccgttatc ggggaagaag tggctgatct
     3601 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat
     3661 gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag
     3721 tccgcagaaa cggaccatga gtggacataa gcctcgttcg gttcgtaagc tgtaatgcaa
     3781 gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg
     3841 cgcagtggc
//
