LOCUS       sequence-75675-e        3644 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 3644)
  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 3644)
  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-MT3T4
FEATURES             Location/Qualifiers
     source          1..3644
                     /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        326..705
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     gap             707..725
                     /estimated_length=19
     primer_bind     complement(756..775)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(757..775)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(782..799)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(782..798)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(791..813)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             936..1238
                     /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     1117..1136
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1587..2246
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1653..1672)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2471..3059
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2960..2979
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      3224..3310
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3402..3429
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     3437..3460
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     promoter        3471..3518
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     3538..3560
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3557..3573
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3557..3573
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3591..3609
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3591..3608
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
       61 ggcaccgagt cggtgctttt ttttgcaaaa ttttccagat cgatttcttc ttcctctgtt
      121 cttcggcgtt caatttctgg ggttttctct tcgttttctg taactgaaac ctaaaatttg
      181 acctaaaaaa aatctcaaat aatatgattc agtggttttg tacttttcag ttagttgagt
      241 tttgcagttc cgatgagata aaccaataag taattcatcc aggtcaccaa gttctaggat
      301 tttcagaact gcaacttatt ttatcaagga atctttaaac atacgaacag atcacttaaa
      361 gttcttctga agcaacttaa agttatcagg catgcatgga tcttggagga atcagatgtg
      421 cagtcaggga ccatagcaca agacaggcgt cttctactgg tgctaccagc aaatgctgga
      481 agccgggaac actgggtacg ttggaaacca cgtgatgtga agaagtaaga taaactgtag
      541 gagaaaagca tttcgtagtg ggccatgaag cctttcagga catgtattgc agtatgggcc
      601 ggcccattac gcaattggac gacaacaaag actagtatta gtaccacctc ggctatccac
      661 atagatcaaa gctgatttaa aagagttgtg cagatgatcc gtggcgnnnn nnnnnnnnnn
      721 nnnnnccttt ggcctatttg gcctggccca attcgcccta tagtgagtcg tattacaatt
      781 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
      841 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
      901 gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac acctataaaa
      961 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgccggggc
     1021 gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
     1081 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcaa
     1141 gtgtgccagt atccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     1201 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg agattatcaa
     1261 aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa cggaccatga
     1321 gtggacataa gcctcgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca
     1381 actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg
     1441 cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc
     1501 gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca
     1561 gtcgccctaa aacaaagtta aacatcatgg agaaaaaaat cactggatat accaccgttg
     1621 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta
     1681 cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaagta
     1741 agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg
     1801 agttccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt
     1861 acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg
     1921 atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg
     1981 cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga
     2041 gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca
     2101 caatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc
     2161 atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact
     2221 gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc gatcctctag
     2281 ctagaaattc gttcaagccg acgccgcttc gcggcgcggc ttaactcaag cgttagatgc
     2341 actaagcaca taattgctca cagccaaact atcaggtcaa gtcccgcgga gttgttcggt
     2401 aaattgtcac aacgccgccc gccccgttcc actgagcgtc agacccggta gaaaagatca
     2461 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2521 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2581 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2641 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2701 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2761 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2821 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2881 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2941 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3001 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3061 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3121 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgctagg atggatctcg
     3181 ggcacgtcta actactaagc gagagtaggg aactgccagg catcaaataa aacgaaaggc
     3241 tcagtcggaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     3301 taggacaaat ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg
     3361 ggcaggacgc ccgccataaa ctgccaggca tcaaactaag cagaaggcca tcctgacgga
     3421 tggccttttt gcgtttctac aaactcttcc tgttagttag ttacttaagt gttgacaatt
     3481 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccgagc
     3541 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct atttaggtga
     3601 cactatagaa tactcaagct atgcaaggcc cttaaggcca aggg
//
