LOCUS       sequence-259006-        3395 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 3395)
  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 3395)
  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..3395
                     /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             425..443
                     /estimated_length=19
     promoter        complement(495..513)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(496..513)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(531..547)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(531..547)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(544..566)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        complement(586..633)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(644..667)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(675..702)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(794..880)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1045..1633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1125..1144)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1858..2517)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2432..2451
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(2866..3168)
                     /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(2968..2987)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3291..3313
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3305..3322
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3306..3322
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3329..3348
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3329..3347
                     /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 caatttcttt aatccaaact actgcagcct gacagacaaa tgaggatgca
      181 aacaatttta aagtttatct aacgctagct gttttgtttc ttctctctgg tgcaccaacg
      241 acggcgtttt ctcaatcata aagaggcttg ttttacttaa ggccaataat gttgatggat
      301 cgaaagaaga gggcttttaa taaacgagcc cgtttaagct gtaaacgatg tcaaaaacat
      361 cccacatcgt tcagttgaaa atagaagctc tgtttatata ttggtagagt cgactaagag
      421 attgnnnnnn nnnnnnnnnn nnngtttaga gaccaataat ccttggcctt aagggccttg
      481 catagcttga gtattctata gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg
      541 tttcctgtgt gaaattgtta tccgctcggt ttagttcctc accttgtcgt attatactat
      601 gccgatatac tatgccgatg attaattgtc aacacttaag taactaacta acaggaagag
      661 tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt tgatgcctgg
      721 cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca acgttcaaat
      781 ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
      841 gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca gttccctact
      901 ctcgcttagt agttagacgt gcccgagatc catcctagcg gtaatacggt tatccacaga
      961 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     1021 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     1081 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1141 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     1201 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     1261 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     1321 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     1381 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     1441 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     1501 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     1561 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     1621 aaaaggatct caagaagatc ctttgatctt ttctaccggg tctgacgctc agtggaacgg
     1681 ggcgggcggc gttgtgacaa tttaccgaac aactccgcgg gacttgacct gatagtttgg
     1741 ctgtgagcaa ttatgtgctt agtgcatcta acgcttgagt taagccgcgc cgcgaagcgg
     1801 cgtcggcttg aacgaatttc tagctagagg atcgcaccaa taactgcctt aaaaaaatta
     1861 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg
     1921 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc
     1981 ttgcgtataa tatttgccca ttgtgaaaac gggggcgaag aagttgtcca tattggccac
     2041 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc
     2101 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata
     2161 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc
     2221 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc
     2281 gtctttcatt gccatacgga actccggatg agcattcatc aggcgggcaa gaatgtgaat
     2341 aaaggccgga taaaacttgt gcttactttt ctttacggtc tttaaaaagg ccgtaatatc
     2401 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc
     2461 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatgat
     2521 gtttaacttt gttttagggc gactgccctg ctgcgtaaca tcgttgctgc tccataacat
     2581 caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca tagactgtac
     2641 cccaaaaaaa cagtcataac aagccatgaa aaccgccact gcgccgttac caccgctgcg
     2701 ttcggtcaag gttctggacc agttgcgtga gcgcatacgc tacttgcatt acagcttacg
     2761 aaccgaacga ggcttatgtc cactcatggt ccgtttctgc ggactggctt tctacgtgaa
     2821 aaggatctag gtgaagatcc tttttgataa tctcatgcct gacatttata ttccccagaa
     2881 catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc
     2941 cccgataacg gatactggca cacttgccat atcggtggtc atcatgcgcc agctttcatc
     3001 cccgatatgc accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact
     3061 ggccaggggg atcaccatcc gtcgccccgg cgtgtcaata atatcactct gtacatccac
     3121 aaacagacga taacggctct ctcttttata ggtgtaaacc ttaaactgcc gtacgtatag
     3181 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     3241 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     3301 acgttgtaaa acgacggcca gtgaattgta atacgactca ctatagggcg aattgggcca
     3361 ggccaaatag gccaaaggaa taatggtctc tattg
//
