LOCUS       sequence-244476-        3528 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 3528)
  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 3528)
  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-DT2T3
FEATURES             Location/Qualifiers
     source          1..3528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        1..76
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(609..627)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(610..627)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(645..661)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(645..661)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(658..680)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        complement(700..747)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(758..781)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(789..816)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(908..994)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1159..1747)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1239..1258)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1972..2631)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2546..2565
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(2980..3282)
                     /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(3082..3101)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3405..3427
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3419..3436
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3420..3436
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3443..3462
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3443..3461
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
       61 ggcaccgagt cggtgctttt ttttggatag aatttcccag cttttttgcg tgtttcagct
      121 ctcatgatcc ttggccaatg ggtgtagtaa attttctgca cattcattgg atggaaaata
      181 atggttttag ctttagggaa taagaaaagt gtataggaag gggatttttg tacaatcaca
      241 tttgaattag gtctttgaaa tgacagggaa tgaggacata tgatgagacg gtcattgttt
      301 tagttccacc acgattatat ttgaaattta cgtgagtgtg agtgagactt gcataagaaa
      361 ataaaatctt tagttgggaa aaaattcaat aatataaatg ggcttgagaa ggaagcgagg
      421 gataggcctt tttctaaaat aggcccattt aagctattaa caatcttcaa aagtaccaca
      481 gcgcttaggt aaagaaagca gctgagttta tatatggtta gagacgaagt agtgattgtt
      541 accgggaaat ctagatcgtt tagagaccaa taatccttgg ccttaagggc cttgcatagc
      601 ttgagtattc tatagtgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct
      661 gtgtgaaatt gttatccgct cggtttagtt cctcaccttg tcgtattata ctatgccgat
      721 atactatgcc gatgattaat tgtcaacact taagtaacta actaacagga agagtttgta
      781 gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc ctggcagttt
      841 atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc aaatccgctc
      901 ccggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg
      961 cccagtcttc cgactgagcc tttcgtttta tttgatgcct ggcagttccc tactctcgct
     1021 tagtagttag acgtgcccga gatccatcct agcggtaata cggttatcca cagaatcagg
     1081 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     1141 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     1201 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     1261 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     1321 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     1381 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     1441 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     1501 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     1561 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     1621 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1681 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     1741 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acggggcggg
     1801 cggcgttgtg acaatttacc gaacaactcc gcgggacttg acctgatagt ttggctgtga
     1861 gcaattatgt gcttagtgca tctaacgctt gagttaagcc gcgccgcgaa gcggcgtcgg
     1921 cttgaacgaa tttctagcta gaggatcgca ccaataactg ccttaaaaaa attacgcccc
     1981 gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc
     2041 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
     2101 ataatatttg cccattgtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa
     2161 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa
     2221 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg
     2281 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg
     2341 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt
     2401 cattgccata cggaactccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc
     2461 cggataaaac ttgtgcttac ttttctttac ggtctttaaa aaggccgtaa tatccagctg
     2521 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     2581 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca tgatgtttaa
     2641 ctttgtttta gggcgactgc cctgctgcgt aacatcgttg ctgctccata acatcaaaca
     2701 tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtaccccaaa
     2761 aaaacagtca taacaagcca tgaaaaccgc cactgcgccg ttaccaccgc tgcgttcggt
     2821 caaggttctg gaccagttgc gtgagcgcat acgctacttg cattacagct tacgaaccga
     2881 acgaggctta tgtccactca tggtccgttt ctgcggactg gctttctacg tgaaaaggat
     2941 ctaggtgaag atcctttttg ataatctcat gcctgacatt tatattcccc agaacatcag
     3001 gttaatggcg tttttgatgt cattttcgcg gtggctgaga tcagccactt cttccccgat
     3061 aacggatact ggcacacttg ccatatcggt ggtcatcatg cgccagcttt catccccgat
     3121 atgcaccacc gggtaaagtt cacgggagac tttatctgac agcagacgtg cactggccag
     3181 ggggatcacc atccgtcgcc ccggcgtgtc aataatatca ctctgtacat ccacaaacag
     3241 acgataacgg ctctctcttt tataggtgta aaccttaaac tgccgtacgt ataggctgcg
     3301 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     3361 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     3421 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gccaggccaa
     3481 ataggccaaa ggaataatgg tctctattgg caaagttata caatggca
//
