LOCUS       sequence-75668-e        3525 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 3525)
  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 3525)
  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..3525
                     /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"
     gap             555..573
                     /estimated_length=19
     promoter        complement(625..643)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(626..643)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(661..677)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(661..677)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(674..696)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        complement(716..763)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(774..797)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(805..832)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(924..1010)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1175..1763)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1255..1274)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1988..2647)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2562..2581
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(2996..3298)
                     /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(3098..3117)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3421..3443
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3435..3452
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3436..3452
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3459..3478
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3459..3477
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
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 aaccaatatt aatccaaact actgcagcct gacagacaaa
      301 tgaggatgca aacaatttta aagtttatct aacgctagct gttttgtttc ttctctctgg
      361 tgcaccaacg acggcgtttt ctcaatcata aagaggcttg ttttacttaa ggccaataat
      421 gttgatggat cgaaagaaga gggcttttaa taaacgagcc cgtttaagct gtaaacgatg
      481 tcaaaaacat cccacatcgt tcagttgaaa atagaagctc tgtttatata ttggtagagt
      541 cgactaagag attgnnnnnn nnnnnnnnnn nnngtttaga gaccaataat ccttggcctt
      601 aagggccttg catagcttga gtattctata gtgtcaccta aatagcttgg cgtaatcatg
      661 gtcatagctg tttcctgtgt gaaattgtta tccgctcggt ttagttcctc accttgtcgt
      721 attatactat gccgatatac tatgccgatg attaattgtc aacacttaag taactaacta
      781 acaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt
      841 tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca
      901 acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
      961 cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca
     1021 gttccctact ctcgcttagt agttagacgt gcccgagatc catcctagcg gtaatacggt
     1081 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     1141 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     1201 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     1261 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     1321 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     1381 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1441 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     1501 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     1561 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     1621 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     1681 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     1741 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctaccggg tctgacgctc
     1801 agtggaacgg ggcgggcggc gttgtgacaa tttaccgaac aactccgcgg gacttgacct
     1861 gatagtttgg ctgtgagcaa ttatgtgctt agtgcatcta acgcttgagt taagccgcgc
     1921 cgcgaagcgg cgtcggcttg aacgaatttc tagctagagg atcgcaccaa taactgcctt
     1981 aaaaaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc
     2041 tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca
     2101 ccttgtcgcc ttgcgtataa tatttgccca ttgtgaaaac gggggcgaag aagttgtcca
     2161 tattggccac gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa
     2221 acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat
     2281 cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg
     2341 aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca
     2401 ccagctcacc gtctttcatt gccatacgga actccggatg agcattcatc aggcgggcaa
     2461 gaatgtgaat aaaggccgga taaaacttgt gcttactttt ctttacggtc tttaaaaagg
     2521 ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct
     2581 caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt
     2641 tctccatgat gtttaacttt gttttagggc gactgccctg ctgcgtaaca tcgttgctgc
     2701 tccataacat caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca
     2761 tagactgtac cccaaaaaaa cagtcataac aagccatgaa aaccgccact gcgccgttac
     2821 caccgctgcg ttcggtcaag gttctggacc agttgcgtga gcgcatacgc tacttgcatt
     2881 acagcttacg aaccgaacga ggcttatgtc cactcatggt ccgtttctgc ggactggctt
     2941 tctacgtgaa aaggatctag gtgaagatcc tttttgataa tctcatgcct gacatttata
     3001 ttccccagaa catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag
     3061 ccacttcttc cccgataacg gatactggca cacttgccat atcggtggtc atcatgcgcc
     3121 agctttcatc cccgatatgc accaccgggt aaagttcacg ggagacttta tctgacagca
     3181 gacgtgcact ggccaggggg atcaccatcc gtcgccccgg cgtgtcaata atatcactct
     3241 gtacatccac aaacagacga taacggctct ctcttttata ggtgtaaacc ttaaactgcc
     3301 gtacgtatag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     3361 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     3421 cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca ctatagggcg
     3481 aattgggcca ggccaaatag gccaaaggaa taatggtctc tattg
//
