LOCUS       sequence-259004-        3586 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 3586)
  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 3586)
  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-MT1T2.2
FEATURES             Location/Qualifiers
     source          1..3586
                     /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             649..667
                     /estimated_length=19
     primer_bind     complement(698..717)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(699..717)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(724..741)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(724..740)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(733..755)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             878..1180
                     /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     1059..1078
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1529..2188
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1595..1614)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2413..3001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2902..2921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      3166..3252
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3344..3371
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     3379..3402
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     promoter        3413..3460
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     3480..3502
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3499..3515
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3499..3515
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3533..3551
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3533..3550
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
       61 ggcaccgagt cggtgctttt ttttttcgtt ttgcattgag ttttctccgt cgcatgtttg
      121 cagcatgaat ccaaaccaca cggagttcaa attcccacag attaaggctc gtccgtcgca
      181 caaggtaatg tgtgaatatt atatctgtcg tgcaaaattg cctggcctgc acaattgctg
      241 ttatagttgg cggcagggag agttttaaca ttgactagcg tgctgataat ttgtgagaaa
      301 taataattga caagtagata ctgacatttg agaagagctt ctgaactgtt attagtaaca
      361 aaaatggaaa gctgatgcac ggaaaaagga aagaaaaagc catacttttt tttaggtagg
      421 aaaagaaaaa gccatacgag actgatgtct ctcagatggg ccgggatctg tctatctagc
      481 aggcagcagc ccaccaacct cacgggccag caattacgag tccttctaaa agctcccgcc
      541 gaggggcgct ggcgctgctg tgcagcagca cgtctaacat tagtcccacc tcgccagttt
      601 acagggagca gaaccagctt ataagcggag gcgcggcacc aagaagcgnn nnnnnnnnnn
      661 nnnnnnncct ttggcctatt tggcctggcc caattcgccc tatagtgagt cgtattacaa
      721 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
      781 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
      841 tcgcccttcc caacagttgc gcagcctata cgtacggcag tttaaggttt acacctataa
      901 aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgccggg
      961 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga
     1021 actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc
     1081 aagtgtgcca gtatccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
     1141 catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggca tgagattatc
     1201 aaaaaggatc ttcacctaga tccttttcac gtagaaagcc agtccgcaga aacggaccat
     1261 gagtggacat aagcctcgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg
     1321 caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat
     1381 ggcttgttat gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc
     1441 gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg
     1501 cagtcgccct aaaacaaagt taaacatcat ggagaaaaaa atcactggat ataccaccgt
     1561 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg
     1621 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaag
     1681 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc
     1741 ggagttccgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg
     1801 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga
     1861 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct
     1921 ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt
     1981 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt
     2041 cacaatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
     2101 tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta
     2161 ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc agttattggt gcgatcctct
     2221 agctagaaat tcgttcaagc cgacgccgct tcgcggcgcg gcttaactca agcgttagat
     2281 gcactaagca cataattgct cacagccaaa ctatcaggtc aagtcccgcg gagttgttcg
     2341 gtaaattgtc acaacgccgc ccgccccgtt ccactgagcg tcagacccgg tagaaaagat
     2401 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2461 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2521 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2581 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2641 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2701 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2761 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2821 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2881 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2941 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3001 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3061 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcta ggatggatct
     3121 cgggcacgtc taactactaa gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     3181 gctcagtcgg aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     3241 agtaggacaa atccgccggg agcggatttg aacgttgtga agcaacggcc cggagggtgg
     3301 cgggcaggac gcccgccata aactgccagg catcaaacta agcagaaggc catcctgacg
     3361 gatggccttt ttgcgtttct acaaactctt cctgttagtt agttacttaa gtgttgacaa
     3421 ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccga
     3481 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctatttaggt
     3541 gacactatag aatactcaag ctatgcaagg cccttaaggc caaggg
//
