LOCUS       sequence-210724-        3544 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 3544)
  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 3544)
  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-DT1T2
FEATURES             Location/Qualifiers
     source          1..3544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        20..95
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(644..662)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(645..662)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(680..696)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(680..696)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(693..715)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        complement(735..782)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(793..816)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(824..851)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(943..1029)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1194..1782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1274..1293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2007..2666)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2581..2600
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(3015..3317)
                     /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(3117..3136)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3440..3462
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3454..3471
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3455..3471
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3478..3497
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3478..3496
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gacggccatg gcggttctcg ttttagagct agaaatagca agttaaaata aggctagtcc
       61 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tttgcaaaat tttccagatc
      121 gatttcttct tcctctgttc ttcggcgttc aatttctggg gttttctctt cgttttctgt
      181 aactgaaacc taaaatttga cctaaaaaaa atctcaaata atatgattca gtggttttgt
      241 acttttcagt tagttgagtt ttgcagttcc gatgagataa accaatatta atccaaacta
      301 ctgcagcctg acagacaaat gaggatgcaa acaattttaa agtttatcta acgctagctg
      361 ttttgtttct tctctctggt gcaccaacga cggcgttttc tcaatcataa agaggcttgt
      421 tttacttaag gccaataatg ttgatggatc gaaagaagag ggcttttaat aaacgagccc
      481 gtttaagctg taaacgatgt caaaaacatc ccacatcgtt cagttgaaaa tagaagctct
      541 gtttatatat tggtagagtc gactaagaga ttggcaaagt tatacaatgg cagtttagag
      601 accaataatc cttggcctta agggccttgc atagcttgag tattctatag tgtcacctaa
      661 atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcggtt
      721 tagttcctca ccttgtcgta ttatactatg ccgatatact atgccgatga ttaattgtca
      781 acacttaagt aactaactaa caggaagagt ttgtagaaac gcaaaaaggc catccgtcag
      841 gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc
      901 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag
      961 agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact gagcctttcg
     1021 ttttatttga tgcctggcag ttccctactc tcgcttagta gttagacgtg cccgagatcc
     1081 atcctagcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1141 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1201 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1261 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1321 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1381 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1441 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1501 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1561 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1621 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1681 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1741 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1801 tctacggggt ctgacgctca gtggaacggg gcgggcggcg ttgtgacaat ttaccgaaca
     1861 actccgcggg acttgacctg atagtttggc tgtgagcaat tatgtgctta gtgcatctaa
     1921 cgcttgagtt aagccgcgcc gcgaagcggc gtcggcttga acgaatttct agctagagga
     1981 tcgcaccaat aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt
     2041 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
     2101 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat tgtgaaaacg
     2161 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
     2221 ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
     2281 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     2341 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     2401 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa ctccggatga
     2461 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttacttttc
     2521 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     2581 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     2641 gtatatccag tgattttttt ctccatgatg tttaactttg ttttagggcg actgccctgc
     2701 tgcgtaacat cgttgctgct ccataacatc aaacatcgac ccacggcgta acgcgcttgc
     2761 tgcttggatg cccgaggcat agactgtacc ccaaaaaaac agtcataaca agccatgaaa
     2821 accgccactg cgccgttacc accgctgcgt tcggtcaagg ttctggacca gttgcgtgag
     2881 cgcatacgct acttgcatta cagcttacga accgaacgag gcttatgtcc actcatggtc
     2941 cgtttctgcg gactggcttt ctacgtgaaa aggatctagg tgaagatcct ttttgataat
     3001 ctcatgcctg acatttatat tccccagaac atcaggttaa tggcgttttt gatgtcattt
     3061 tcgcggtggc tgagatcagc cacttcttcc ccgataacgg atactggcac acttgccata
     3121 tcggtggtca tcatgcgcca gctttcatcc ccgatatgca ccaccgggta aagttcacgg
     3181 gagactttat ctgacagcag acgtgcactg gccaggggga tcaccatccg tcgccccggc
     3241 gtgtcaataa tatcactctg tacatccaca aacagacgat aacggctctc tcttttatag
     3301 gtgtaaacct taaactgccg tacgtatagg ctgcgcaact gttgggaagg gcgatcggtg
     3361 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     3421 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa
     3481 tacgactcac tatagggcga attgggccag gccaaatagg ccaaaggaat aatggtctct
     3541 attg
//
