LOCUS       sequence-307547-       10362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR system used for genome editing in Mycobacterium tuberculosis.
            Helper plasmid expresses Sth1 Cas9 and the cognate sgRNA, using 
            hygromycin as a selection marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10362)
  AUTHORS   Yan MY, Li SS, Ding XY, Guo XP, Jin Q, Sun YC
  TITLE     A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient 
            Genome Editing in Mycobacterium tuberculosis.
  JOURNAL   mBio. 2020 Jan 28;11(1). pii: mBio.02364-19. doi: 
            10.1128/mBio.02364-19.
  PUBMED    31992616
REFERENCE   2  (bases 1 to 10362)
  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: "mBio."; 
            date: "2020-01-28"; volume: "11(1). pii: mBio.02364-19. doi"; pages:
            " 10.1128/mBio.02364-19"
COMMENT     Sequence Label: pYC2085
FEATURES             Location/Qualifiers
     source          1..10362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1004..1031)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    4511..4529
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4541..4559
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      4765..5353
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5254..5273
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(8502..8521)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 acactggcag agcattacgc tgacttgacg ggacggcggc tttgttgaat aaatcgaact
       61 tttgctgagt tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca
      121 aagcaaaagt tcaaaatcac caactggtcc acctacaaca aagctctcac caaccgtggc
      181 tccctcactt tctggctgga tgatggggcg attcaggcct ggtatgagtc agcaacacct
      241 tcttcacgag gcagacctca ctagttatca cgaaccgctt tcgcacttca gttgcttttc
      301 cagcccgcag atgatcagct ccagcccgaa caaaaaggcg ggttcggcgc cctggtggtc
      361 aaacagttcg atcgcctggc gcaacaacgg gggcatcgaa tccgtcgtgg gggtctcccg
      421 ttcctctttc gccacctgat gttcctgatc ctccaacacg cagcccaagg tgaagtgtcc
      481 cacggcggac agggcataca gggcgttctc caacgaaaaa ccctgttgac agaggaaggc
      541 cagctggttc tccagggtct cgtattgctt ctcggtcggg cgggtgccca agtggacttt
      601 ggcgccatcc cgatggctca ggagggcgca ccggaacgac ttcgcgttgt tccgcagaaa
      661 atcctgccag ctctcgccct ccagggggca aaagtgcgtg tgatggcgat ccagcatctc
      721 gatggccaac gcatccagca gcgcgcgctt gttcttcacg tgccagtaga gggtcggttg
      781 ttccacgccg agcttttggg ccaacttgcg cgtggtcagt ccctcgatac cgacctcgtt
      841 cagcagctcc agcgccgagt tgatgacctt ggacttatcc aaccgcgaca tgcgaagtcc
      901 tcctgatctg acagattata tcaaccgagc acacgcatcg caatctatca cttaattaaa
      961 aaaaaaaaag cgccgcagtt gcggcgcttt ttttttttct agaaaaaggc catccgtcag
     1021 gatggccttc tgcttagcta atcagcggcc gcacgcgttg cgctcggtcg ttcggctgcg
     1081 gcgagcggta tcagctcact caaaggcggt aaagaaaaat ggccagagta tttagaagtc
     1141 cagcttcggc ttgtcgccct cgttcttgat gatgtgctgg ttgcccagca cgtcggtgcg
     1201 caccttgtag atcgagatgt tcgacttgcc caggcccttc ttgcactggc ccgagttggc
     1261 cacgttgccc agcaccttga tcagggcctc gccgccctcg aacttctgct tgtcgtacgg
     1321 cttcagctcc acgtagtgct tctgcttcgg catggtgcgc gacaggaagc ggaacagctg
     1381 ctgctccttg gtctcggtgt ccttcaccag cagcaggtcg ttcttgtaca gggtgaactt
     1441 gaactccgag tccgagtcca cgccctcctt cttcttgatg tcgttgtact tctcctgcga
     1501 gatcttgtag gtgccggtgc ccttgtcgaa ctgcaggtcg gcgtacttca ggcccaggat
     1561 ctcgtacttg ccggtggtct tgttgaagta cacgtcggcg cgccacggcg acaccgactg
     1621 cagcaccacc ttgttgttcg agtccttcgg ggtgatgtcg atgtggttgc ccagcttcga
     1681 gtcgtagtac ttcagcgact tgatctccgg gccgttgccc ttcttcgagt acttgcggat
     1741 gtagccgtgc tcctccttgt acttcaggaa cgggttgcac ggcacctcct tgcccttgtc
     1801 gttgatctgc ttgttcgggt agttctccag gatcggctcg atcaccttct cgaaggtctg
     1861 cgggtcgtgg cggtacatca ggaacttcga cttgtccttc ttgtagatct tcatgaaggc
     1921 gtcgtagccg tcctgggtgt agatgtcctt gatcttgccc agcacgtagg tctcgtcggc
     1981 cttgtccttg cccaccttgg cctggcgggt ggcgtagatg gtggcgtccg agatcttgcg
     2041 gttgaacttc gagtccacct ggtacgagaa caggatcgag tcctcgaact ccttcgactt
     2101 cagggtgtcc acgaagtgct ggtacggggc cttgaacacc gactccttgt actcgtcgtc
     2161 cgagatcagc tcgccggtct cgatgtccag cagctggtcc tccgagtacg acaccagggt
     2221 gttcttctgc ttcttccaca ggttcagctg cgacgaggcg gcgatgatca gggcgtccac
     2281 ggcgtggtgg tggtaggtgt cgcgggtctt ttcgatgccc cagtggcggc gcagctgcga
     2341 ggtgaactgg ccgcgcacca ccgacacctt ggtgtcgatc ttgtgggcgc ggaagtgctc
     2401 ctgcagggcg ttcagcacca cgcgcgaggc gtagcgggtg tccaccaggt tgcgctcgat
     2461 gaacttcttg cgcacgtcga acttcgagat gtcctcctcg gtcagcaggt actccttctt
     2521 cttgttcgac agggtcttgc tctcgcgcac gaaggccttc agctcgcgga acgaccaggc
     2581 gtcgtccatc gagtccaggg cctggtacgg ggtgcgctgg cccttctcct ggttggcggt
     2641 ggcgtacacc agcaccttgt tggccagcga gtcgtcgaag gtgatcgaca gcggcaggat
     2701 gtggtccacc tcgaactggt tcgagttgtt gatcaggtcg tggatcgaga tggtcttgcc
     2761 ggtgtacagg cagcgctcgc cctgctggtg ccacaggcgg atcttggtgg ccagctgctt
     2821 gtggccgtgg aacaccgagt gcggcagctc ggccttgccg ttgtactggt tggcggcctt
     2881 cagcatggcg gcgtccttct cgtccttgtt ggccttctgg atcttctgga tggccttctt
     2941 ctcgtcgtcc tcgttggtct cgcgggccat ctcgatcacg atgttgtcga agtcgccgta
     3001 ctccttgatg gcggcgttca cgatcttgat ggcctggcgc accgacttgg ccaccaccgg
     3061 gttgtagatc tcctcggtca gcagcttctc gtcgatgtac ttggtcttgt tcgacgacga
     3121 ggtggtcttt tgcttgccca ggcgggtcag gatggtcatc tgctcctccg aggtctcgta
     3181 cagctccggg atcagctcca tcatcagctt caccgagaag ttgtgccagc ccttgccgaa
     3241 gatcgacgag ttggccttgc ggaactgcac cagctcgtcc acctgcttct gcgagaacga
     3301 gccgtcggcg aactcgtgct ccagggcctc ctggatgccc tcgcgctcgg tgttcagggt
     3361 cagcacgtag gccagcttgt ccagggtctc gcggtccatc tgctcgatgt ccagggtctc
     3421 cagggttttc atcttgcggt aggcctcgaa ggtgtggatc tcggccttgc ccgacttgtc
     3481 gatgcggtag cccttgatgt cggccacgtc gcacgacagc agcttggcga tgtacttgaa
     3541 cagcttggcc gggcccatgg ccttctcgtt cttcacgtag ttgatgatct ggttcttctg
     3601 ctccttcgac agcttcttgg tctcggtcgg cacggtcagg ttgttcaggt cgttcagcag
     3661 gttgaactcc tgggcggtgt acgaggcctt ggcggcgcgg aactcgtccg ggtagaaggt
     3721 gcacttgccg atcaggatgc cgaagatgtt gtccagggtc tcgcccgagg tgcggtagcg
     3781 gccgtagtcg gtgcgcgact tctcgttgcc cgggccgtgg tagtacttgc gcttgccggt
     3841 caggatctcc aggtagcggt tgatgaactc gtcggtgatc tgcgggttga actcctgctg
     3901 ggtctgcagg atgcgcaggg cctccgagcg gtaggccgag gtcgggaaca cgttgatcag
     3961 gcggtgcttc ttgccgtcct tctccacggt gaagtcgccg cgcagctggc cgtaggtctg
     4021 gtagcgctcc agctggatct ggcccggggt cttggtctcc agctgcttcg agttctcctt
     4081 cacgatctgg gcgtagtcgc ccaccgacga gttgccgtcg tccgaggcgt cgtccaggta
     4141 cgagatgccg cggtgcttca ccatgttctt cagggcgatg aacagctcct cgttcgacag
     4201 ctcgtcggtc aggcccttca cgcgcagctg gtacgggttc aggttgatcg agatcttggt
     4261 gaagtcggtg atcaggcccg actcctcgaa caggcggttc aggcgcacgc ggcggtgctt
     4321 cttgcggcgg gccaggcggc ggccctggcg gttggtgcgg cgcaccaggt tgttctcggc
     4381 ctgggcggcc gggaagatgc gcgagttctt gtggatgatc tcgccggtca ccttgttcag
     4441 gatgcccacg cccaccgagc cgatgccgat gtccaggccc agcaccaggt ccgacatatc
     4501 gataccgcct tctctatcac tgatagggac tcccagatta tctctatcac tgatagggaa
     4561 gtcaaatccc caggtcagag ggctattttc cctggtcaga catatgaaaa aaaaaagccc
     4621 cgcaatcgcg gggctttttt ttttcaaaca acagataaac gaaaggccca gtctttcgac
     4681 tgagcctttc gtttattgtt ccatgagcgt cagaccacta gttccatgag cgtcagaccc
     4741 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4801 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4861 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4921 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4981 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5041 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5101 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5161 agaaagcgcc acgcttcccg aggggagaaa ggcggacagg tatccggtaa gcggcagggt
     5221 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5281 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5341 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5401 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5461 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcaacg cgtgagccca
     5521 ccagctccgt aagttcgggc gctgtgtggc tcgtacccgc gcattcaggc ggcagggggt
     5581 ctaacgggtc taaggcggcg tgtacggccg ccacagcggc tctcagcggc ccggaaacgt
     5641 cctcgaaacg acgcatgtgt tcctcctggt tggtacaggt ggttgggggt gctcggctgt
     5701 cgctggtgtt ccaccaccag ggctcgacgg gagagcgggg gagtgtgcag ttgggagccg
     5761 gatgggtagt tggtcccgtc gggtaggggt gctccattgg gccagcgtat tggggaccac
     5821 cgacaaaatc acgcgccgaa cgcccccaac tcccaagtca gacaacgcct tcagatggaa
     5881 agtgttcgcg cagtgcctgg cgttgacttc tttgtctcgc cggcagtcat gccggggtga
     5941 actcggccga aaaccgagca ccgcttcggc gtgtcggtac caggtacagg actttctctg
     6001 ggggacgatc gtcggcatgg cccaaccgca taaggggcag cgcgagcaga tcaaggtccg
     6061 cgctgccgca gtcgtgtacg cgcgtctgcg cgaaatggcc gcccaacgcg gcaccagcgt
     6121 cagccagctc tcggcagacc tcctcgccat tgcagtcggt catcccgagg ccgtccgcga
     6181 actcgacaag gaggtgctgc cgctggcgat gtgagagctc gcgctgaggc cccgccctgc
     6241 tcgactgcgg gggccttggc agatgtgaca tctcaatagc tggtgtgcgg attcgcccgc
     6301 tgacatgacg aaggcccccg aaggggcctt gtctgaaggt atcgacctga gcgccaactc
     6361 aggtccgggt ccgcacccgt tgcccctgaa agggacgtgt cttggtcgac tcgtctcacg
     6421 gtagcgcatg cctgcccggc gtaccagata agccacgcag caagtgcgcg aatcgtgatc
     6481 ttctcaccgc ccgaccgagt catcgggcgt gttcttcacc gccaccgctc ggcgcggcgg
     6541 cctatgccgg acacagcccg cacgtcgagc gcgcccgggt ggcacagggt cggcgtgact
     6601 gggtgctgcg cgccggtgcg aaacatcgcg cgccgatgtg gacgagtcgc gtgtcatggt
     6661 tggacggtct gcggcggtgg gcgcagtcgc cggcgctggg cgagttgtgt gcggagcagc
     6721 gggtgtcgat gaccgcggcc acgttgttgg cgatcgcggc ggtgatggcc gagcacgccg
     6781 atcacgccac cggccgccac gtggcggtca ctcgcgccac catcgccgac acagtgggat
     6841 gtgacgtgcg caccgtgacc gcggcatggc gggtgctgcg ggtgtcgcgc tgggcggtgg
     6901 aggcacagcg cggccacggc tcgccaacca cccccagcgt gggccggcga ccctcggtgt
     6961 accacctcgt gccgcgtcgc gccccccagc cggccggtga tcagcctgtg cacgatttcc
     7021 acctaccgcc gccaggcggc gatggttttt taactcccgt agggagtaag tcaccaagcg
     7081 tgcgcgcacg cgcacacgcc acctatagcc gcgaacaaca acccgatcca ccgcggcgct
     7141 ggcgcaccac accgcgacca cttgccatcc agttgctcgc cgcccagctc gtcgcgcgca
     7201 cccacggcct gggccgcggc cacatcggcg ccatctgcga cacgcttacc gccgcgggca
     7261 tcgagccggc cgtctggtcg gcacgagcta tcaccgacgc cctcgacgcc gacatgcgca
     7321 cccgcggatg cagctggccc gaccacatca ccaaccccgc agcgtttctg tccagccggc
     7381 tacgacggct ctcgtggacc acgccgccag caccgccaaa aggcggcggc tacgccgcca
     7441 cccgcatcga ccagacgccg gcccgggcga cgctcaccga cgcagcgcag gcccgcattg
     7501 ccgccgcccg cgaacacatc cgccaagtgc ttaccaaccg cgctcgacgc ccccgccctg
     7561 agcgcactcc gagcgcgtgt tcagacgcac cgcaacgatc tatcgtcgaa ggcatggcac
     7621 cgtccactcc caacgaggcg attccgccgt caacgccggt gcgccgctgg cggcacgagg
     7681 agtgcaccgt gcacgacaaa ctgcccgcag ggcgtatcta ctgagttact gatctactga
     7741 tagaatgcgt tcatgaccga tgaaggaccg cagagcggta gccagatgag cgcggctgaa
     7801 cactggcggg cgtggctaga acgctacggc gacgattacg ccacggagcg caattgggga
     7861 gcgggtgtcg cgggggttcc gtggggggtt ccgttgcaac gggtcggaca ggtaaaagtc
     7921 ctggtagacg ctagttttct ggtttgggcc atgcctgtct cgttgcgtgt ttcgttgcgc
     7981 ccgttttgaa taccagccag acgagacggg gttctacgaa tcttggtcga taccaagcca
     8041 tttccgctga atatcgggga gctcaccgcc agaatcggtg gttgtggtga tgtacgtggc
     8101 gaactccgtt gtagtgcctg tggtggcatc cgtggcgcgg ccgcgggatc ctctgaccag
     8161 ggaaaatagc cctctgacct ggggatttgc gatccctatc agtgatagat ataatctggg
     8221 agtgtcttca ttaggaagac atgtttttgt actcgaaaga agctacaaag ataaggcttc
     8281 atgccgaaat caacaccctg tcattttatg gcagggtgtt ttttttttgt cgacttgggg
     8341 accctagagg tccccttttt tatttttttg gatgaagagc attatcaatg gctcttcaca
     8401 gggctagcaa aaaaaaaagc cccgcgattg cggggctttt tttttttacg gttatccctc
     8461 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     8521 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccc atgcgccaga
     8581 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     8641 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     8701 tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga
     8761 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     8821 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     8881 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     8941 tggctggcct gttgaacaag tctggaaaga aatgcataat cttttgccat tctcaccgga
     9001 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     9061 aattcaggcg ccgggggcgg tgtccggcgg cccccagagg aactgcgcca gttcctccgg
     9121 atcggtgaag ccggagagat ccagcggggt ctcctcgaac acctcgaagt cgtgcaggaa
     9181 ggtgaaggcg agcagttcgc gggcgaagtc ctcggtccgc ttccactgcg ccccgtcgag
     9241 cagcgcggcc aggatctcgc ggtcgccccg gaaggcgttg agatgcagtt gcaccaggct
     9301 gtagcgggag tctcccgcat agacgtcggt gaagtcgacg atcccggtga cctcggtcgc
     9361 ggccaggtcc acgaagatgt tggtcccgtg caggtcgccg tggacgaacc ggggttcgcg
     9421 gccggccagc agcgtgtcca cgtccggcag ccagtcctcc aggcggtcca gcagccgggg
     9481 cgagaggtag ccccacccgc ggtggtcctc gacggtcgcc gcgcggcgtt cccgcagcag
     9541 ttccgggaac acctcgctat ggggggtgag cacggtgttc ccggtcagcg gcaccctgtg
     9601 cagccggccg agcacccggc cgagttcgcg ggccagggcg agcagcgcgt tccggtcggt
     9661 cgtgccgtcc atcgcggacc gccaggtggt gccggtcatc cggctcatca ccaggtaggg
     9721 ccacggccag gctccggtgc cgggccgcag ctcgccgcgg ccgaggaggc ggggcaccgg
     9781 caccggggcg tccgccagga ccgcgtacgc ctccgactcc gacgcgaggc tctccggacc
     9841 gcaccagtgc tcgccgaaca gcttgatcac cgggtcgggc tcgccgacca gtacggggtt
     9901 ggtgctctcg ccgggcaccc gcagcaccgg cggcaccggc agcccgagct cctccagggc
     9961 tcggcgggcc agcggctccc agaattcctg gtcgttccgc aggctcgcgt aggaatcatc
    10021 cgaatcaata cggtcgagaa gtaacaggga ttcttgtgtc acggtgaatc ctcctgaata
    10081 tgtagagctt ctttcctgca ctatgatgag tgcaccccgc tcaagcttgc ctcgacggcg
    10141 gtgtgctgtc aattcagagc tgagcctgat gcactcaact tactgagcat gctaacgctg
    10201 gtcgtgcggg tcggaattat ggaactgcct cggtgagttt tctccttcat tacagaaacg
    10261 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
    10321 gctcgatgag tttttctaat cagaattggt taattggttg ta
//
