LOCUS       sequence-266367-        3364 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR template for sgRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3364)
  AUTHORS   Zhang Q, Zhang Y, Lu MH, Chai YP, Jiang YY, Zhou Y, Wang XC, Chen QJ
  TITLE     A Novel Ternary Vector System United with Morphogenic Genes Enhances
            CRISPR/Cas Delivery in Maize.
  JOURNAL   Plant Physiol. 2019 Sep 26. pii: pp.19.00767. doi: 
            10.1104/pp.19.00767.
  PUBMED    31558579
REFERENCE   2  (bases 1 to 3364)
  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: "Plant 
            Physiol. 2019 Sep 26. pii: pp.19.00767. doi: 10.1104/pp.19.00767."
COMMENT     Sequence Label: pCBC-DT1T2.2
FEATURES             Location/Qualifiers
     source          1..3364
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..48
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     68..90
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     87..103
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     87..103
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        121..139
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     121..138
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_RNA        195..270
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(650..669)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(651..669)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(676..693)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(676..692)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(685..707)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             830..1132
                     /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     1011..1030
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1481..2140
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1547..1566)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2365..2953
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2854..2873
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      3118..3204
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3296..3323
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     3331..3354
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
ORIGIN
        1 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
       61 taaaccgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
      121 atttaggtga cactatagaa tactcaagct atgcaaggcc cttaaggcca aggtccactg
      181 agcgtcaggt ctcagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
      241 caacttgaaa aagtggcacc gagtcggtgc ttttttttgc aaaattttcc agatcgattt
      301 cttcttcctc tgttcttcgg cgttcaattt ctttaatcca aactactgca gcctgacaga
      361 caaatgagga tgcaaacaat tttaaagttt atctaacgct agctgttttg tttcttctct
      421 ctggtgcacc aacgacggcg ttttctcaat cataaagagg cttgttttac ttaaggccaa
      481 taatgttgat ggatcgaaag aagagggctt ttaataaacg agcccgttta agctgtaaac
      541 gatgtcaaaa acatcccaca tcgttcagtt gaaaatagaa gctctgttta tatattggta
      601 gagtcgacta agagattggc ctttggccta tttggcctgg cccaattcgc cctatagtga
      661 gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
      721 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
      781 ggcccgcacc gatcgccctt cccaacagtt gcgcagccta tacgtacggc agtttaaggt
      841 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat
      901 tgacacgccg gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa
      961 agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac
     1021 caccgatatg gcaagtgtgc cagtatccgt tatcggggaa gaagtggctg atctcagcca
     1081 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg
     1141 catgagatta tcaaaaagga tcttcaccta gatccttttc acgtagaaag ccagtccgca
     1201 gaaacggacc atgagtggac ataagcctcg ttcggttcgt aagctgtaat gcaagtagcg
     1261 tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt
     1321 ggcggttttc atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc
     1381 caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt
     1441 tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatc atggagaaaa aaatcactgg
     1501 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
     1561 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
     1621 cgtaaagaaa agtaagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
     1681 gaatgctcat ccggagttcc gtatggcaat gaaagacggt gagctggtga tatgggatag
     1741 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
     1801 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
     1861 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc
     1921 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
     1981 cgcccccgtt ttcacaatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
     2041 ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga
     2101 attacaacag tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg
     2161 gtgcgatcct ctagctagaa attcgttcaa gccgacgccg cttcgcggcg cggcttaact
     2221 caagcgttag atgcactaag cacataattg ctcacagcca aactatcagg tcaagtcccg
     2281 cggagttgtt cggtaaattg tcacaacgcc gcccgccccg ttccactgag cgtcagaccc
     2341 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2401 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2461 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     2521 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2581 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2641 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2701 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2761 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2821 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2881 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2941 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3001 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3061 taggatggat ctcgggcacg tctaactact aagcgagagt agggaactgc caggcatcaa
     3121 ataaaacgaa aggctcagtc ggaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     3181 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgt gaagcaacgg
     3241 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaac taagcagaag
     3301 gccatcctga cggatggcct ttttgcgttt ctacaaactc ttcctgttag ttagttactt
     3361 aagt
//
