LOCUS       62056_11895        5757 bp DNA     circular SYN 09-AUG-2020
DEFINITION  Cloning vector pgRNA-ccdB-2, complete sequence.
ACCESSION   MN934324
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5757)
  AUTHORS   Zhang K, Zhang Z, Kang J, Chen J, Liu J, Gao N, Fan L, Zheng P, Wang
            Y, Sun J.
  TITLE     CRISPR/Cas13d-Mediated Microbial RNA Knockdown
  JOURNAL   Front Bioeng Biotechnol 8, 856 (2020)
REFERENCE   2  (bases 1 to 5757)
  AUTHORS   Wang Y.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-JAN-2020) Key Laboratory of Systems Microbial 
            Biotechnology, Tianjin Institute of Industrial Biotechnology, 
            Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport 
            Economic Area, Tianjin 300308, China
REFERENCE   3  (bases 1 to 5757)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Front 
            Bioeng Biotechnol 8, 856 (2020)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (13-JAN-2020) Key Laboratory of Systems Microbial Biotechnology, 
            Tianjin Institute of Industrial Biotechnology, Chinese Academy of 
            Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin
            300308, China"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5757
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1..53
                     /regulatory_class="promoter"
     misc_RNA        54..83
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d
                     gRNA (Konermann et al., 2018)"
     CDS             432..734
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
     terminator      753..839
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      931..958
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     rep_origin      complement(2055..3518)
                     /direction=LEFT
     CDS             4042..4833
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      4936..5524
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ttttctccac ataagctggc aatgttgcga cgcaacaggt acagtgtaat tcaaacccct
       61 accaactggt cggggtttga aactgagacc acgcgtggat ccggcttact aaaagccaga
      121 taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac tgatatgtat
      181 acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg acagttgaca
      241 gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtaagcac
      301 aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga
      361 agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg acgagaacag
      421 gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt
      481 ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg
      541 ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg
      601 gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggta tccgttatcg
      661 gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga
      721 tgttctgggg aatataaggt ctcaccaggc atcaaataaa acgaaaggct cagtcgaaag
      781 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
      841 cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc
      901 cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg
      961 cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1021 aattaattcc gctagatgac gtgcggcttc gacctcctgg gcgtggcgct tgttggcgcg
     1081 ctcgcggctg gctgcggcac gacacgcgtc tgagcagtat tttgcgcgcc gtcctcgtgg
     1141 gtcaggccgg ggtgggatca ggccaccgca gtaggcgcag ctgatgcgat cctccactac
     1201 tgcgcgtcct cctggcgctg ccgagcacgc agctcgtcgg ccagctcttc aaggtcggcc
     1261 acaagcgttt ctaggtcgct cgcggcactt gcccagtcgc gtgatgctgg cgcgtctgtc
     1321 gtatcgaggg cgcggaaaaa tccgatcacc gtttttaaat cgacggcggc atcgagtgcg
     1381 tcggactcca gcgcgacatc ggagagatcc accgctgatg cttcaggcca gttttggtac
     1441 ttcgtcgtga aggtcatgac accattataa cgaacgttcg ttaaaaattc tagccccaat
     1501 tctgataatt tcttccggca ctcctgcgaa aacctgcgag acttcttgcc cagaaaaaac
     1561 gccaagcgca gcggttaccg cacttttttt ccaggtgatt tcaccctgac cagcgaagcg
     1621 gcactttagt gcatgaggtg tgcccctggt ttcccctctt tggagggttc aacccaaaaa
     1681 agcacacaag caaaaatgaa aatcatcatg agcaagttgg tgcgaagcag caacgcgcta
     1741 gctccaaaaa ggtctgcagg atctcgagga gatttttgag ggggagggag tcgaggaaga
     1801 gccagagcag aaggcgggga accgttctct gccgacagcg tgagcccccc ttaaaaatca
     1861 ggccggggag gaaccgggga gggatcagag ctaggagcga gacaccctaa agggggggaa
     1921 ccgttttctg ctgacggtgt ttcgtttatt agttttcagc ccgtggatag cggagggtga
     1981 gggcaagtga gagccagagc aaggacggga cccctaaagg ggggaaccgt tttctgctga
     2041 cggtgtttcg tttattagtt ttcagcccgt ggacggccgc gtttagcttc cattccaagt
     2101 gcctttctga cttgttggat gcgcctttca ctgacaccta gttcgcctgc aagctcacga
     2161 gtcgagggat cagcaaccga ttgagaacgg gcatccagga tcgcagtttt gacgcgaagt
     2221 tcgagcaact cgcctgtcat ttctcggcgt ttgtttgctt ccgctaatcg ctgtcgcgtc
     2281 tcctgcgcat acttactttc tgggtcagcc catctgcgtg cattcgatgt agctgcgccc
     2341 cgtcgcccca tcgtcgctag agctttccgc cctcggctgc tctgcgtttc cacccgacga
     2401 gcagggacga ctggctggcc tttagccacg tagccgcgca cacgacgcgc catcgtcagg
     2461 cgatcacgca tggcgggaag atccggctcc cggccgtctg caccgaccgc ctgggcaacg
     2521 ttgtacgcca cttcatacgc gtcgatgatc ttggcatctt ttaggcgctc accagcagct
     2581 ttgagctggt atcccacggt caacgcgtgg cgaaacgcgg tttcgtcgcg cgctcgctct
     2641 ggatttgtcc agagcactcg cacgccgtcg atcaggtcgc cggacgcgtc cagggcgctc
     2701 ggcaggctcg cgtccaaaat cgctagcgcc ttggcttctg cggtggcgcg ttgtgccgct
     2761 tcaatgcggg cgcgtccgct ggaaaagtcc tgctcaatgt actttttcgg cttctgtgat
     2821 ccggtcatcg ttcgagcaat ctccattagg tcggccagcc gatccacacg atcatgctgg
     2881 cagtgccatt tataggctgt cggatcgtct gagacgtgca gcggccaccg gctcagccta
     2941 tgcgaaaaag cctggtcagc gccgaaaaca cgagtcattt cttccgtcgt tgcagccagc
     3001 aggcgcatat ttgggctggt tttacctgct gcggcataca ccgggtcaat gagccagatg
     3061 agctggcatt tcccgctcag cggattcacg ccgatccaag ccggcgcttt ttctaggcgt
     3121 gcccatttct ctaaaatcgc gtagacctgc gggtttacgt gctcaatctt cccgccggcc
     3181 tggtggctgg gcacatcgat gtcaagcacg atcaccgcgg catgttgcgc gtgcgtcagc
     3241 gcaacgtact ggcaccgcgt cagcgctttt gagccagccc ggtagagctt tggttgggtt
     3301 tcgccggtat ccgggttttt aatccaggcg ctcgcgaaat ctcttgtctt gctgccctgg
     3361 aagctttcgc gtcccaggtg agcgagcagt tcgcggcgat cttctgccgt ccagccgcgt
     3421 gagccgcagc gcatagcttc ggggtgggtg tcgaacagat cggcggacaa tttccacgcg
     3481 ctagctgtga ctgtgtcctg cggatcggct agagtcatgt cttgagtgct ttctcccagc
     3541 tgatgactgg gggttagccg acgccctgtg agttcccgct cacggggcgt tcaacttttt
     3601 caggtatttg tgcagcttat cgtgttttct tcgtaaatga acgcttaact accttgttaa
     3661 acgtggcaaa taggcaggat tgatggggat ctagcttcac gctgccgcaa gcactcaggg
     3721 cgcaagggct gctaaaggaa gcggtagaaa gccagtccgc agaaacggtg ctgaccccgg
     3781 atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag
     3841 gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc
     3901 gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac
     3961 tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag
     4021 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4081 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4141 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4201 tccggtgccc tgaatgaact ccaagacgag gcagcgcggc tatcgtggct ggccacgacg
     4261 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4321 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4381 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4441 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4501 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4561 ctcaaggcgc ggatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4621 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4681 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4741 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4801 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgcggaat
     4861 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4921 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4981 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5041 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     5101 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5161 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5221 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5281 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5341 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5401 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5461 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5521 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5581 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     5641 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     5701 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccg
//
