LOCUS       sequence-266369-        3605 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 3605)
  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 3605)
  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..3605
                     /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        194..269
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(891..910)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(892..910)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(917..934)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(917..933)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(926..948)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1071..1373
                     /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     1252..1271
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1722..2381
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1788..1807)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2606..3194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3095..3114
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      3359..3445
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3537..3564
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     3572..3595
                     /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 aggggcttcg
      181 tgccaaccaa cgagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
      241 aacttgaaaa agtggcaccg agtcggtgct tttttttttc gttttgcatt gagttttctc
      301 cgtcgcatgt ttgcagcatg aatccaaacc acacggagtt caaattccca cagattaagg
      361 ctcgtccgtc gcacaaggta atgtgtgaat attatatctg tcgtgcaaaa ttgcctggcc
      421 tgcacaattg ctgttatagt tggcggcagg gagagtttta acattgacta gcgtgctgat
      481 aatttgtgag aaataataat tgacaagtag atactgacat ttgagaagag cttctgaact
      541 gttattagta acaaaaatgg aaagctgatg cacggaaaaa ggaaagaaaa agccatactt
      601 ttttttaggt aggaaaagaa aaagccatac gagactgatg tctctcagat gggccgggat
      661 ctgtctatct agcaggcagc agcccaccaa cctcacgggc cagcaattac gagtccttct
      721 aaaagctccc gccgaggggc gctggcgctg ctgtgcagca gcacgtctaa cattagtccc
      781 acctcgccag tttacaggga gcagaaccag cttataagcg gaggcgcggc accaagaagc
      841 gtgaccagga ggagcatgcc cctttggcct atttggcctg gcccaattcg ccctatagtg
      901 agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
      961 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     1021 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct atacgtacgg cagtttaagg
     1081 tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta
     1141 ttgacacgcc ggggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata
     1201 aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga
     1261 ccaccgatat ggcaagtgtg ccagtatccg ttatcgggga agaagtggct gatctcagcc
     1321 accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag
     1381 gcatgagatt atcaaaaagg atcttcacct agatcctttt cacgtagaaa gccagtccgc
     1441 agaaacggac catgagtgga cataagcctc gttcggttcg taagctgtaa tgcaagtagc
     1501 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
     1561 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
     1621 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     1681 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat catggagaaa aaaatcactg
     1741 gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt
     1801 cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga
     1861 ccgtaaagaa aagtaagcac aagttttatc cggcctttat tcacattctt gcccgcctga
     1921 tgaatgctca tccggagttc cgtatggcaa tgaaagacgg tgagctggtg atatgggata
     1981 gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga
     2041 gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt
     2101 acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag
     2161 ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct
     2221 tcgcccccgt tttcacaatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc
     2281 tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg
     2341 aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa ggcagttatt
     2401 ggtgcgatcc tctagctaga aattcgttca agccgacgcc gcttcgcggc gcggcttaac
     2461 tcaagcgtta gatgcactaa gcacataatt gctcacagcc aaactatcag gtcaagtccc
     2521 gcggagttgt tcggtaaatt gtcacaacgc cgcccgcccc gttccactga gcgtcagacc
     2581 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2641 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2701 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2761 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2821 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2881 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2941 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3001 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3061 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3121 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3181 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3241 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3301 ctaggatgga tctcgggcac gtctaactac taagcgagag tagggaactg ccaggcatca
     3361 aataaaacga aaggctcagt cggaagactg ggcctttcgt tttatctgtt gtttgtcggt
     3421 gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg tgaagcaacg
     3481 gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ctaagcagaa
     3541 ggccatcctg acggatggcc tttttgcgtt tctacaaact cttcctgtta gttagttact
     3601 taagt
//
