LOCUS       sequence-320843-       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"
     protein_bind    1..19
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    31..49
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      3529..3556
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     6401..6420
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(9569..10157)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9649..9668)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tccctatcag tgatagagat aatctgggag tccctatcag tgatagagaa ggcggtatcg
       61 atatgtcgga cctggtgctg ggcctggaca tcggcatcgg ctcggtgggc gtgggcatcc
      121 tgaacaaggt gaccggcgag atcatccaca agaactcgcg catcttcccg gccgcccagg
      181 ccgagaacaa cctggtgcgc cgcaccaacc gccagggccg ccgcctggcc cgccgcaaga
      241 agcaccgccg cgtgcgcctg aaccgcctgt tcgaggagtc gggcctgatc accgacttca
      301 ccaagatctc gatcaacctg aacccgtacc agctgcgcgt gaagggcctg accgacgagc
      361 tgtcgaacga ggagctgttc atcgccctga agaacatggt gaagcaccgc ggcatctcgt
      421 acctggacga cgcctcggac gacggcaact cgtcggtggg cgactacgcc cagatcgtga
      481 aggagaactc gaagcagctg gagaccaaga ccccgggcca gatccagctg gagcgctacc
      541 agacctacgg ccagctgcgc ggcgacttca ccgtggagaa ggacggcaag aagcaccgcc
      601 tgatcaacgt gttcccgacc tcggcctacc gctcggaggc cctgcgcatc ctgcagaccc
      661 agcaggagtt caacccgcag atcaccgacg agttcatcaa ccgctacctg gagatcctga
      721 ccggcaagcg caagtactac cacggcccgg gcaacgagaa gtcgcgcacc gactacggcc
      781 gctaccgcac ctcgggcgag accctggaca acatcttcgg catcctgatc ggcaagtgca
      841 ccttctaccc ggacgagttc cgcgccgcca aggcctcgta caccgcccag gagttcaacc
      901 tgctgaacga cctgaacaac ctgaccgtgc cgaccgagac caagaagctg tcgaaggagc
      961 agaagaacca gatcatcaac tacgtgaaga acgagaaggc catgggcccg gccaagctgt
     1021 tcaagtacat cgccaagctg ctgtcgtgcg acgtggccga catcaagggc taccgcatcg
     1081 acaagtcggg caaggccgag atccacacct tcgaggccta ccgcaagatg aaaaccctgg
     1141 agaccctgga catcgagcag atggaccgcg agaccctgga caagctggcc tacgtgctga
     1201 ccctgaacac cgagcgcgag ggcatccagg aggccctgga gcacgagttc gccgacggct
     1261 cgttctcgca gaagcaggtg gacgagctgg tgcagttccg caaggccaac tcgtcgatct
     1321 tcggcaaggg ctggcacaac ttctcggtga agctgatgat ggagctgatc ccggagctgt
     1381 acgagacctc ggaggagcag atgaccatcc tgacccgcct gggcaagcaa aagaccacct
     1441 cgtcgtcgaa caagaccaag tacatcgacg agaagctgct gaccgaggag atctacaacc
     1501 cggtggtggc caagtcggtg cgccaggcca tcaagatcgt gaacgccgcc atcaaggagt
     1561 acggcgactt cgacaacatc gtgatcgaga tggcccgcga gaccaacgag gacgacgaga
     1621 agaaggccat ccagaagatc cagaaggcca acaaggacga gaaggacgcc gccatgctga
     1681 aggccgccaa ccagtacaac ggcaaggccg agctgccgca ctcggtgttc cacggccaca
     1741 agcagctggc caccaagatc cgcctgtggc accagcaggg cgagcgctgc ctgtacaccg
     1801 gcaagaccat ctcgatccac gacctgatca acaactcgaa ccagttcgag gtggaccaca
     1861 tcctgccgct gtcgatcacc ttcgacgact cgctggccaa caaggtgctg gtgtacgcca
     1921 ccgccaacca ggagaagggc cagcgcaccc cgtaccaggc cctggactcg atggacgacg
     1981 cctggtcgtt ccgcgagctg aaggccttcg tgcgcgagag caagaccctg tcgaacaaga
     2041 agaaggagta cctgctgacc gaggaggaca tctcgaagtt cgacgtgcgc aagaagttca
     2101 tcgagcgcaa cctggtggac acccgctacg cctcgcgcgt ggtgctgaac gccctgcagg
     2161 agcacttccg cgcccacaag atcgacacca aggtgtcggt ggtgcgcggc cagttcacct
     2221 cgcagctgcg ccgccactgg ggcatcgaaa agacccgcga cacctaccac caccacgccg
     2281 tggacgccct gatcatcgcc gcctcgtcgc agctgaacct gtggaagaag cagaagaaca
     2341 ccctggtgtc gtactcggag gaccagctgc tggacatcga gaccggcgag ctgatctcgg
     2401 acgacgagta caaggagtcg gtgttcaagg ccccgtacca gcacttcgtg gacaccctga
     2461 agtcgaagga gttcgaggac tcgatcctgt tctcgtacca ggtggactcg aagttcaacc
     2521 gcaagatctc ggacgccacc atctacgcca cccgccaggc caaggtgggc aaggacaagg
     2581 ccgacgagac ctacgtgctg ggcaagatca aggacatcta cacccaggac ggctacgacg
     2641 ccttcatgaa gatctacaag aaggacaagt cgaagttcct gatgtaccgc cacgacccgc
     2701 agaccttcga gaaggtgatc gagccgatcc tggagaacta cccgaacaag cagatcaacg
     2761 acaagggcaa ggaggtgccg tgcaacccgt tcctgaagta caaggaggag cacggctaca
     2821 tccgcaagta ctcgaagaag ggcaacggcc cggagatcaa gtcgctgaag tactacgact
     2881 cgaagctggg caaccacatc gacatcaccc cgaaggactc gaacaacaag gtggtgctgc
     2941 agtcggtgtc gccgtggcgc gccgacgtgt acttcaacaa gaccaccggc aagtacgaga
     3001 tcctgggcct gaagtacgcc gacctgcagt tcgacaaggg caccggcacc tacaagatct
     3061 cgcaggagaa gtacaacgac atcaagaaga aggagggcgt ggactcggac tcggagttca
     3121 agttcaccct gtacaagaac gacctgctgc tggtgaagga caccgagacc aaggagcagc
     3181 agctgttccg cttcctgtcg cgcaccatgc cgaagcagaa gcactacgtg gagctgaagc
     3241 cgtacgacaa gcagaagttc gagggcggcg aggccctgat caaggtgctg ggcaacgtgg
     3301 ccaactcggg ccagtgcaag aagggcctgg gcaagtcgaa catctcgatc tacaaggtgc
     3361 gcaccgacgt gctgggcaac cagcacatca tcaagaacga gggcgacaag ccgaagctgg
     3421 acttctaaat actctggcca tttttcttta ccgcctttga gtgagctgat accgctcgcc
     3481 gcagccgaac gaccgagcgc aacgcgtgcg gccgctgatt agctaagcag aaggccatcc
     3541 tgacggatgg cctttttcta gaaaaaaaaa aagcgccgca actgcggcgc tttttttttt
     3601 ttaattaagt gatagattgc gatgcgtgtg ctcggttgat ataatctgtc agatcaggag
     3661 gacttcgcat gtcgcggttg gataagtcca aggtcatcaa ctcggcgctg gagctgctga
     3721 acgaggtcgg tatcgaggga ctgaccacgc gcaagttggc ccaaaagctc ggcgtggaac
     3781 aaccgaccct ctactggcac gtgaagaaca agcgcgcgct gctggatgcg ttggccatcg
     3841 agatgctgga tcgccatcac acgcactttt gccccctgga gggcgagagc tggcaggatt
     3901 ttctgcggaa caacgcgaag tcgttccggt gcgccctcct gagccatcgg gatggcgcca
     3961 aagtccactt gggcacccgc ccgaccgaga agcaatacga gaccctggag aaccagctgg
     4021 ccttcctctg tcaacagggt ttttcgttgg agaacgccct gtatgccctg tccgccgtgg
     4081 gacacttcac cttgggctgc gtgttggagg atcaggaaca tcaggtggcg aaagaggaac
     4141 gggagacccc cacgacggat tcgatgcccc cgttgttgcg ccaggcgatc gaactgtttg
     4201 accaccaggg cgccgaaccc gcctttttgt tcgggctgga gctgatcatc tgcgggctgg
     4261 aaaagcaact gaagtgcgaa agcggttcgt gataactagt gaggtctgcc tcgtgaagaa
     4321 ggtgttgctg actcatacca ggcctgaatc gccccatcat ccagccagaa agtgagggag
     4381 ccacggttgg tgagagcttt gttgtaggtg gaccagttgg tgattttgaa cttttgcttt
     4441 gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa
     4501 gttcgattta ttcaacaaag ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt
     4561 acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt
     4621 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     4681 aaaactcacc gaggcagttc cataattccg acccgcacga ccagcgttag catgctcagt
     4741 aagttgagtg catcaggctc agctctgaat tgacagcaca ccgccgtcga ggcaagcttg
     4801 agcggggtgc actcatcata gtgcaggaaa gaagctctac atattcagga ggattcaccg
     4861 tgacacaaga atccctgtta cttctcgacc gtattgattc ggatgattcc tacgcgagcc
     4921 tgcggaacga ccaggaattc tgggagccgc tggcccgccg agccctggag gagctcgggc
     4981 tgccggtgcc gccggtgctg cgggtgcccg gcgagagcac caaccccgta ctggtcggcg
     5041 agcccgaccc ggtgatcaag ctgttcggcg agcactggtg cggtccggag agcctcgcgt
     5101 cggagtcgga ggcgtacgcg gtcctggcgg acgccccggt gccggtgccc cgcctcctcg
     5161 gccgcggcga gctgcggccc ggcaccggag cctggccgtg gccctacctg gtgatgagcc
     5221 ggatgaccgg caccacctgg cggtccgcga tggacggcac gaccgaccgg aacgcgctgc
     5281 tcgccctggc ccgcgaactc ggccgggtgc tcggccggct gcacagggtg ccgctgaccg
     5341 ggaacaccgt gctcaccccc catagcgagg tgttcccgga actgctgcgg gaacgccgcg
     5401 cggcgaccgt cgaggaccac cgcgggtggg gctacctctc gccccggctg ctggaccgcc
     5461 tggaggactg gctgccggac gtggacacgc tgctggccgg ccgcgaaccc cggttcgtcc
     5521 acggcgacct gcacgggacc aacatcttcg tggacctggc cgcgaccgag gtcaccggga
     5581 tcgtcgactt caccgacgtc tatgcgggag actcccgcta cagcctggtg caactgcatc
     5641 tcaacgcctt ccggggcgac cgcgagatcc tggccgcgct gctcgacggg gcgcagtgga
     5701 agcggaccga ggacttcgcc cgcgaactgc tcgccttcac cttcctgcac gacttcgagg
     5761 tgttcgagga gaccccgctg gatctctccg gcttcaccga tccggaggaa ctggcgcagt
     5821 tcctctgggg gccgccggac accgcccccg gcgcctgaat taatttcccc tcgtcaaaaa
     5881 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     5941 gattatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     6001 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     6061 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     6121 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     6181 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     6241 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     6301 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tggggcttcc
     6361 catacaagcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     6421 catataaatc agcatccatg ttggaattta atcgcggcct cgagggataa ccgtaaaaaa
     6481 aaaaagcccc gcaatcgcgg ggcttttttt tttgctagcc ctgtgaagag ccattgataa
     6541 tgctcttcat ccaaaaaaat aaaaaagggg acctctaggg tccccaagtc gacaaaaaaa
     6601 aaacaccctg ccataaaatg acagggtgtt gatttcggca tgaagcctta tctttgtagc
     6661 ttctttcgag tacaaaaaca tgtcttccta atgaagacac tcccagatta tatctatcac
     6721 tgatagggat cgcaaatccc caggtcagag ggctattttc cctggtcaga ggatcccgcg
     6781 gccgcgccac ggatgccacc acaggcacta caacggagtt cgccacgtac atcaccacaa
     6841 ccaccgattc tggcggtgag ctccccgata ttcagcggaa atggcttggt atcgaccaag
     6901 attcgtagaa ccccgtctcg tctggctggt attcaaaacg ggcgcaacga aacacgcaac
     6961 gagacaggca tggcccaaac cagaaaacta gcgtctacca ggacttttac ctgtccgacc
     7021 cgttgcaacg gaacccccca cggaaccccc gcgacacccg ctccccaatt gcgctccgtg
     7081 gcgtaatcgt cgccgtagcg ttctagccac gcccgccagt gttcagccgc gctcatctgg
     7141 ctaccgctct gcggtccttc atcggtcatg aacgcattct atcagtagat cagtaactca
     7201 gtagatacgc cctgcgggca gtttgtcgtg cacggtgcac tcctcgtgcc gccagcggcg
     7261 caccggcgtt gacggcggaa tcgcctcgtt gggagtggac ggtgccatgc cttcgacgat
     7321 agatcgttgc ggtgcgtctg aacacgcgct cggagtgcgc tcagggcggg ggcgtcgagc
     7381 gcggttggta agcacttggc ggatgtgttc gcgggcggcg gcaatgcggg cctgcgctgc
     7441 gtcggtgagc gtcgcccggg ccggcgtctg gtcgatgcgg gtggcggcgt agccgccgcc
     7501 ttttggcggt gctggcggcg tggtccacga gagccgtcgt agccggctgg acagaaacgc
     7561 tgcggggttg gtgatgtggt cgggccagct gcatccgcgg gtgcgcatgt cggcgtcgag
     7621 ggcgtcggtg atagctcgtg ccgaccagac ggccggctcg atgcccgcgg cggtaagcgt
     7681 gtcgcagatg gcgccgatgt ggccgcggcc caggccgtgg gtgcgcgcga cgagctgggc
     7741 ggcgagcaac tggatggcaa gtggtcgcgg tgtggtgcgc cagcgccgcg gtggatcggg
     7801 ttgttgttcg cggctatagg tggcgtgtgc gcgtgcgcgc acgcttggtg acttactccc
     7861 tacgggagtt aaaaaaccat cgccgcctgg cggcggtagg tggaaatcgt gcacaggctg
     7921 atcaccggcc ggctgggggg cgcgacgcgg cacgaggtgg tacaccgagg gtcgccggcc
     7981 cacgctgggg gtggttggcg agccgtggcc gcgctgtgcc tccaccgccc agcgcgacac
     8041 ccgcagcacc cgccatgccg cggtcacggt gcgcacgtca catcccactg tgtcggcgat
     8101 ggtggcgcga gtgaccgcca cgtggcggcc ggtggcgtga tcggcgtgct cggccatcac
     8161 cgccgcgatc gccaacaacg tggccgcggt catcgacacc cgctgctccg cacacaactc
     8221 gcccagcgcc ggcgactgcg cccaccgccg cagaccgtcc aaccatgaca cgcgactcgt
     8281 ccacatcggc gcgcgatgtt tcgcaccggc gcgcagcacc cagtcacgcc gaccctgtgc
     8341 cacccgggcg cgctcgacgt gcgggctgtg tccggcatag gccgccgcgc cgagcggtgg
     8401 cggtgaagaa cacgcccgat gactcggtcg ggcggtgaga agatcacgat tcgcgcactt
     8461 gctgcgtggc ttatctggta cgccgggcag gcatgcgcta ccgtgagacg agtcgaccaa
     8521 gacacgtccc tttcaggggc aacgggtgcg gacccggacc tgagttggcg ctcaggtcga
     8581 taccttcaga caaggcccct tcgggggcct tcgtcatgtc agcgggcgaa tccgcacacc
     8641 agctattgag atgtcacatc tgccaaggcc cccgcagtcg agcagggcgg ggcctcagcg
     8701 cgagctctca catcgccagc ggcagcacct ccttgtcgag ttcgcggacg gcctcgggat
     8761 gaccgactgc aatggcgagg aggtctgccg agagctggct gacgctggtg ccgcgttggg
     8821 cggccatttc gcgcagacgc gcgtacacga ctgcggcagc gcggaccttg atctgctcgc
     8881 gctgcccctt atgcggttgg gccatgccga cgatcgtccc ccagagaaag tcctgtacct
     8941 ggtaccgaca cgccgaagcg gtgctcggtt ttcggccgag ttcaccccgg catgactgcc
     9001 ggcgagacaa agaagtcaac gccaggcact gcgcgaacac tttccatctg aaggcgttgt
     9061 ctgacttggg agttgggggc gttcggcgcg tgattttgtc ggtggtcccc aatacgctgg
     9121 cccaatggag cacccctacc cgacgggacc aactacccat ccggctccca actgcacact
     9181 cccccgctct cccgtcgagc cctggtggtg gaacaccagc gacagccgag cacccccaac
     9241 cacctgtacc aaccaggagg aacacatgcg tcgtttcgag gacgtttccg ggccgctgag
     9301 agccgctgtg gcggccgtac acgccgcctt agacccgtta gaccccctgc cgcctgaatg
     9361 cgcgggtacg agccacacag cgcccgaact tacggagctg gtgggctcac gcgttgcgct
     9421 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     9481 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     9541 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     9601 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     9661 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     9721 acctgtccgc ctttctcccc tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     9781 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     9841 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     9901 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     9961 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
    10021 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
    10081 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
    10141 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcatggaa
    10201 ctagtggtct gacgctcatg gaacaataaa cgaaaggctc agtcgaaaga ctgggccttt
    10261 cgtttatctg ttgtttgaaa aaaaaaagcc ccgcgattgc ggggcttttt tttttcatat
    10321 gtctgaccag ggaaaatagc cctctgacct ggggatttga ct
//
