LOCUS       62056_4920        7902 bp DNA     circular SYN 09-AUG-2020
DEFINITION  Cloning vector pCasRx-3, complete sequence.
ACCESSION   MN934322
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7902)
  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 7902)
  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 7902)
  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..7902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..19
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      55..67
                     /regulatory_class="ribosome_binding_site"
     CDS             68..2968
                     /label=RfxCas13d
                     /note="RNA-targeting Type VI CRISPR protein from
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
     terminator      3078..3105
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        complement(3274..3329)
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     CDS             3345..3968
                     /label=TetR
                     /note="tetracycline repressor TetR"
     rep_origin      5086..5308
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5356..6303
                     /label=Rep101
                     /note="RepA protein needed for replication with the pSC101 
                     origin"
     promoter        6518..6621
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     CDS             6622..7278
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     terminator      7292..7351
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E.
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     terminator      complement(7411..7440)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     primer_bind     complement(7506..7522)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(7559..7577)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7598..7614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7634..7655)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
ORIGIN
        1 tccctatcag tgatagagat tgacatccct atcagtgata gagatactga gcacaaaggc
       61 acccgatatg atcgaaaaga agaagtcatt tgcaaagggc atgggagtaa aatcaacact
      121 tgtatccggt tcaaaggtat acatgacgac gttcgcagaa ggaagcgatg ccagacttga
      181 aaagatcgtt gaaggcgatt ctatcagatc tgtcaacgaa ggagaagcgt tctcagctga
      241 aatggctgat aagaatgcag gctacaagat cggtaacgca aagttcagcc acccaaaggg
      301 ctatgctgta gttgcaaaca accccttata caccggaccg gtacagcagg atatgctcgg
      361 tctgaaggaa acgcttgaaa agagatattt tggagagtct gccgacggaa atgataatat
      421 ctgtattcag gtcatccata atatcctcga tatcgaaaag atcctcgctg aatatataac
      481 caatgctgct tatgcggtaa acaatatttc cggtcttgat aaggatatca tcggttttgg
      541 taagttcagt acggtctata cttatgatga gttcaaggat cctgaacatc acagagcagc
      601 tttcaacaat aacgataagt taattaatgc catcaaggca cagtatgatg aatttgacaa
      661 tttccttgat aatcctcgtc tcggctactt tggacaggct tttttcagta aggaaggcag
      721 aaattacatt atcaattacg gcaacgagtg ttatgatatt cttgctttac tcagcggatt
      781 gcgtcactgg gtagtacata ataatgagga agaatcaagg atttcccgta catggcttta
      841 taatctcgac aagaatcttg acaacgaata tatctctact ctcaattatc tgtatgatag
      901 aattacaaac gaattaacaa attccttctc aaagaatagt gcagccaacg taaactatat
      961 cgctgaaacc cttggtatta atcctgctga atttgcagag cagtatttca gattcagtat
     1021 catgaaggaa cagaagaatc tcggtttcaa tattactaag ctgagagaag taatgcttga
     1081 cagaaaggat atgtctgaga tccgtaaaaa tcataaggtc tttgattcaa tccgtactaa
     1141 ggtctatact atgatggatt tcgttatcta cagatattac attgaagagg atgcaaaggt
     1201 tgctgctgcc aacaagtctc tgccggataa cgaaaaaagc ctcagtgaaa aggatatctt
     1261 tgttataaat ctcagaggaa gctttaacga tgatcagaag gatgcccttt attatgatga
     1321 ggccaatcgt atttggagaa agctcgaaaa cattatgcac aatatcaagg aattcagagg
     1381 caataagaca cgtgaataca agaagaagga tgctccaaga ctccccagaa ttcttcctgc
     1441 cggaagggat gtttccgcgt tctcaaagtt gatgtacgct cttaccatgt tccttgatgg
     1501 taaggagatc aatgatcttc tcaccacgct catcaataag ttcgataaca tccagagttt
     1561 cctcaaggta atgcctctta tcggagtgaa tgcaaagttt gttgaggaat atgccttctt
     1621 caaggacagc gcaaagattg ctgacgaact caggctgatt aagagctttg ccagaatggg
     1681 agaacctatc gcagatgcaa gacgtgctat gtatatcgat gctatcagga ttctcggaac
     1741 aaacctcagc tatgatgagc ttaaggccct tgccgatact ttttcgcttg atgaaaacgg
     1801 caacaagctt aagaagggca agcacggcat gagaaacttc atcattaata atgtaatcag
     1861 taacaagcgc ttccattatc tcattcgtta cggtgatcct gcacatctcc atgagatcgc
     1921 caagaatgaa gctgttgtaa agttcgtcct cggcaggata gctgatatcc agaagaagca
     1981 gggacagaac ggaaagaatc agatcgacag gtactatgag acctgtatcg gcaaggacaa
     2041 gggcaagtct gtctccgaaa aggttgatgc cctcacaaag attatcaccg gtatgaacta
     2101 cgatcagttc gataagaaga gaagcgttat tgaggatact ggaagagaaa acgctgagag
     2161 agaaaagttc aagaagatca tcagcctcta tcttactgtc atttatcaca tccttaagaa
     2221 tattgttaat atcaatgcgc gttacgttat cggcttccat tgcgttgagc gtgatgcaca
     2281 gctctataag gaaaagggct atgatatcaa cctcaagaag ctcgaagaaa aggggttttc
     2341 atcagtcaca aagctgtgtg caggtattga tgagactgct cctgacaagc gtaaggatgt
     2401 tgaaaaggaa atggctgagc gtgcaaagga atctatcgat agccttgaat ctgcaaatcc
     2461 taagctttac gcaaactata tcaagtattc tgacgagaag aaggctgagg aatttactag
     2521 acagatcaac cgtgagaagg caaagaccgc tctgaatgca tatctcagaa atactaagtg
     2581 gaatgtgata atcagggaag atcttcttag aatcgataat aagacatgta cgctctttag
     2641 aaataaggcc gttcatcttg aagttgcaag atatgttcat gcatatatca acgatattgc
     2701 cgaagtaaac agctatttcc agctttatca ttacatcatg cagagaatca tcatgaacga
     2761 aagatatgaa aagtcttctg gaaaggtaag cgaatacttc gatgctgtga acgatgaaaa
     2821 gaagtacaac gacaggcttc tgaagctgtt gtgcgttcca tttggttact gcatcccgag
     2881 attcaagaat ctctccattg aagctttgtt cgacaggaac gaagcagcta agtttgacaa
     2941 ggaaaagaag aaagtatcag gtaattcata gctgagtagg acaaatccgc cgggagcgga
     3001 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     3061 caggcatcaa attaagcaga aggccatcct gacggatggc cttttgtcga ccatatgcac
     3121 agatgaaaac ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg ctgttttggc
     3181 ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag cgggtcgact
     3241 ctagaggatc tactagtcat atggatttca cttttctcta tcactgatag ggagtggtaa
     3301 aataactcta tcaatgatag agtgtcaaca aaaattagga attaatgatg tctagattag
     3361 ataaaagtaa agtgattaac agcgcattag agctgcttaa tgaggtcgga atcgaaggtt
     3421 taacaacccg taaactcgcc cagaagctag gtgtagagca gcctacattg tattggcatg
     3481 taaaaaataa gcgggctttg ctcgacgcct tagccattga gatgttagat aggcaccata
     3541 ctcacttttg ccctttagaa ggggaaagct ggcaagattt tttacgtaat aacgctaaaa
     3601 gttttagatg tgctttacta agtcatcgcg atggagcaaa agtacattta ggtacacggc
     3661 ctacagaaaa acagtatgaa actctcgaaa atcaattagc ctttttatgc caacaaggtt
     3721 tttcactaga gaatgcatta tatgcactca gcgctgtggg gcattttact ttaggttgcg
     3781 tattggaaga tcaagagcat caagtcgcta aagaagaaag ggaaacacct actactgata
     3841 gtatgccgcc attattacga caagctatcg aattatttga tcaccaaggt gcagagccag
     3901 ccttcttatt cggccttgaa ttgatcatat gcggattaga aaaacaactt aaatgtgaaa
     3961 gtgggtctta aaagcataaa acagaatttg cctggccggt tatccacaga atcaggggat
     4021 aacgcaggaa agaacatgtg agatctctac gggtcggatt tgaagtcgtc ttggtaggag
     4081 gcagcctgaa tggcgaatgc cgatgccctt gagagccttc aacccagtca gctccttccg
     4141 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     4201 cgtaggacag ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     4261 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     4321 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     4381 tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct
     4441 tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg caggccatgc
     4501 tgtccaggca ggtagatgac gaccatcagg gacagcttca aggatcgctc gcggctctta
     4561 ccagcctaac ttcgatcatt ggaccgctga tcgtcacggc gatttatgcc gcctcggcga
     4621 gcacatggaa cgggttggca tggattgtag gcgccgccct ataccttgtc tgcctccccg
     4681 cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct
     4741 cgctaacgga ttcaccactc cgcagacccg ccataaaacg ccctgagaag cccgtgacgg
     4801 gcttttcttg tattatgggt agtttccttg catgaatcca taaaaggcgc ctgtagtgcc
     4861 atttaccccc attcactgcc agagccgtga gcgcagcgaa ctgaatgtca cgaaaaagac
     4921 agcgactcag gtgcctgatg gtcggagaca aaaggaatat tcagcgattt gcccgagctt
     4981 gcgagggtgc tacttaagcc tttagggttt taaggtctgt tttgtagagg agcaaacagc
     5041 gtttgcgaca tccttttgta atactgcgga actgactaaa gtagtgagtt atacacaggg
     5101 ctgggatcta ttctttttat ctttttttat tctttcttta ttctataaat tataaccact
     5161 tgaatataaa caaaaaaaac acacaaaggt ctagcggaat ttacagaggg tctagcagaa
     5221 tttacaagtt ttccagcaaa ggtctagcag aatttacaga tacccacaac tcaaaggaaa
     5281 aggactagta attatcattg actagcccat ctcaattggt atagtgatta aaatcaccta
     5341 gaccaattga gatgtatgtc tgaattagtt gttttcaaag caaatgaact agcgattagt
     5401 cgctatgact taacggagca tgaaaccaag ctaattttat gctgtgtggc actactcaac
     5461 cccacgattg aaaaccctac aaggaaagaa cggacggtat cgttcactta taaccaatac
     5521 gctcagatga tgaacatcag tagggaaaat gcttatggtg tattagctaa agcaaccaga
     5581 gagctgatga cgagaactgt ggaaatcagg aatcctttgg ttaaaggctt tgagattttc
     5641 cagtggacaa actatgccaa gttctcaagc gaaaaattag aattagtttt tagtgaagag
     5701 atattgcctt atcttttcca gttaaaaaaa ttcataaaat ataatctgga acatgttaag
     5761 tcttttgaaa acaaatactc tatgaggatt tatgagtggt tattaaaaga actaacacaa
     5821 aagaaaactc acaaggcaaa tatagagatt agccttgatg aatttaagtt catgttaatg
     5881 cttgaaaata actaccatga gtttaaaagg cttaaccaat gggttttgaa accaataagt
     5941 aaagatttaa acacttacag caatatgaaa ttggtggttg ataagcgagg ccgcccgact
     6001 gatacgttga ttttccaagt tgaactagat agacaaatgg atctcgtaac cgaacttgag
     6061 aacaaccaga taaaaatgaa tggtgacaaa ataccaacaa ccattacatc agattcctac
     6121 ctacataacg gactaagaaa aacactacac gatgctttaa ctgcaaaaat tcagctcacc
     6181 agttttgagg caaaattttt gagtgacatg caaagtaagt atgatctcaa tggttcgttc
     6241 tcatggctca cgcaaaaaca acgaaccaca ctagagaaca tactggctaa atacggaagg
     6301 atctgaggtt cttatggctc ttgtatctat cagtgaagca tcaagactaa caaacaaaag
     6361 tagaacaact gttcaccgtt acatatcaaa gggaaaactg tccatatgca cagatgaaaa
     6421 cggtgtaaaa aagatagata catcagagct tttacgagtt tttggtgcat ttaaagctgt
     6481 tcaccatgaa cagatcgaca atgtaactac tagaggttga tcgggcacgt aagaggttcc
     6541 aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt atcgagattt
     6601 tcaggagcta aggaagctaa aatggagaaa aaaatcacgg gatataccac cgttgatata
     6661 tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat
     6721 aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac
     6781 aagttttatc cggcctttat tcacattctt gcccgcctga tgaacgctca cccggagttt
     6841 cgtatggcca tgaaagacgg tgagctggtg atctgggata gtgttcaccc ttgttacacc
     6901 gttttccatg agcaaactga aacgttttcg tccctctgga gtgaatacca cgacgatttc
     6961 cggcagtttc tccacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
     7021 ttccctaaag ggtttattga gaatatgttt tttgtctcag ccaatccctg ggtgagtttc
     7081 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcacgatg
     7141 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgatcca ggttcatcat
     7201 gccgtttgtg atggcttcca tgtcggccgc atgcttaatg aattacaaca gtactgtgat
     7261 gagtggcagg gcggggcgta ataatactag ctccggcaaa aaaacgggca aggtgtcacc
     7321 accctgccct ttttctttaa aaccgaaaag attacttcgc gtttgccacc tgacgtctaa
     7381 gaaaaggaat attcagcaat ttgcccgtgc cgaagaaagg cccacccgtg aaggtgagcc
     7441 agtgagttga ttgctacgta attagttagt tagcccttag tgactcgaat tcctgcagcc
     7501 cgggggatcc actagttcta gagcggccgc caccgcggtg gagctccagc ttttgttccc
     7561 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgag
     7621 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     7681 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     7741 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     7801 ttcggctgcg gcgagcggta tcagctcact caaaggcgcg gttctggcaa atattctggt
     7861 tcaccgttac atatcaaagg gaaaactgtc catatgaagc tt
//
