LOCUS       sequence-320840-        9993 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 
            zeocin as a selection marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9993)
  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 9993)
  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: pYC1640
FEATURES             Location/Qualifiers
     source          1..9993
                     /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"
     CDS             4860..5234
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    5298..5432
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5335..5354)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5389..5408
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6032..6051
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(9200..9788)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9280..9299)
                     /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 ggattcacca
     4861 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
     4921 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     4981 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     5041 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     5101 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     5161 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     5221 aggagcagga ctgaccgacg ccgaccaaca ccgccggtcc gacgcggccc gacgggtccg
     5281 aggggggtcg acctcgaaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5341 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5401 aactcatcaa tgtatcttat catgtctgga tcatggcaag atcctggtat cggtctgcga
     5461 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     5521 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agattatgca
     5581 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     5641 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     5701 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     5761 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     5821 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     5881 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     5941 caacgctacc tttgccatgt ttcagaaaca actctggcgc atggggcttc ccatacaagc
     6001 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     6061 cagcatccat gttggaattt aatcgcggcc tcgagggata accgtaaaaa aaaaaagccc
     6121 cgcaatcgcg gggctttttt ttttgctagc cctgtgaaga gccattgata atgctcttca
     6181 tccaaaaaaa taaaaaaggg gacctctagg gtccccaagt cgacaaaaaa aaaacaccct
     6241 gccataaaat gacagggtgt tgatttcggc atgaagcctt atctttgtag cttctttcga
     6301 gtacaaaaac atgtcttcct aatgaagaca ctcccagatt atatctatca ctgataggga
     6361 tcgcaaatcc ccaggtcaga gggctatttt ccctggtcag aggatcccgc ggccgcgcca
     6421 cggatgccac cacaggcact acaacggagt tcgccacgta catcaccaca accaccgatt
     6481 ctggcggtga gctccccgat attcagcgga aatggcttgg tatcgaccaa gattcgtaga
     6541 accccgtctc gtctggctgg tattcaaaac gggcgcaacg aaacacgcaa cgagacaggc
     6601 atggcccaaa ccagaaaact agcgtctacc aggactttta cctgtccgac ccgttgcaac
     6661 ggaacccccc acggaacccc cgcgacaccc gctccccaat tgcgctccgt ggcgtaatcg
     6721 tcgccgtagc gttctagcca cgcccgccag tgttcagccg cgctcatctg gctaccgctc
     6781 tgcggtcctt catcggtcat gaacgcattc tatcagtaga tcagtaactc agtagatacg
     6841 ccctgcgggc agtttgtcgt gcacggtgca ctcctcgtgc cgccagcggc gcaccggcgt
     6901 tgacggcgga atcgcctcgt tgggagtgga cggtgccatg ccttcgacga tagatcgttg
     6961 cggtgcgtct gaacacgcgc tcggagtgcg ctcagggcgg gggcgtcgag cgcggttggt
     7021 aagcacttgg cggatgtgtt cgcgggcggc ggcaatgcgg gcctgcgctg cgtcggtgag
     7081 cgtcgcccgg gccggcgtct ggtcgatgcg ggtggcggcg tagccgccgc cttttggcgg
     7141 tgctggcggc gtggtccacg agagccgtcg tagccggctg gacagaaacg ctgcggggtt
     7201 ggtgatgtgg tcgggccagc tgcatccgcg ggtgcgcatg tcggcgtcga gggcgtcggt
     7261 gatagctcgt gccgaccaga cggccggctc gatgcccgcg gcggtaagcg tgtcgcagat
     7321 ggcgccgatg tggccgcggc ccaggccgtg ggtgcgcgcg acgagctggg cggcgagcaa
     7381 ctggatggca agtggtcgcg gtgtggtgcg ccagcgccgc ggtggatcgg gttgttgttc
     7441 gcggctatag gtggcgtgtg cgcgtgcgcg cacgcttggt gacttactcc ctacgggagt
     7501 taaaaaacca tcgccgcctg gcggcggtag gtggaaatcg tgcacaggct gatcaccggc
     7561 cggctggggg gcgcgacgcg gcacgaggtg gtacaccgag ggtcgccggc ccacgctggg
     7621 ggtggttggc gagccgtggc cgcgctgtgc ctccaccgcc cagcgcgaca cccgcagcac
     7681 ccgccatgcc gcggtcacgg tgcgcacgtc acatcccact gtgtcggcga tggtggcgcg
     7741 agtgaccgcc acgtggcggc cggtggcgtg atcggcgtgc tcggccatca ccgccgcgat
     7801 cgccaacaac gtggccgcgg tcatcgacac ccgctgctcc gcacacaact cgcccagcgc
     7861 cggcgactgc gcccaccgcc gcagaccgtc caaccatgac acgcgactcg tccacatcgg
     7921 cgcgcgatgt ttcgcaccgg cgcgcagcac ccagtcacgc cgaccctgtg ccacccgggc
     7981 gcgctcgacg tgcgggctgt gtccggcata ggccgccgcg ccgagcggtg gcggtgaaga
     8041 acacgcccga tgactcggtc gggcggtgag aagatcacga ttcgcgcact tgctgcgtgg
     8101 cttatctggt acgccgggca ggcatgcgct accgtgagac gagtcgacca agacacgtcc
     8161 ctttcagggg caacgggtgc ggacccggac ctgagttggc gctcaggtcg ataccttcag
     8221 acaaggcccc ttcgggggcc ttcgtcatgt cagcgggcga atccgcacac cagctattga
     8281 gatgtcacat ctgccaaggc ccccgcagtc gagcagggcg gggcctcagc gcgagctctc
     8341 acatcgccag cggcagcacc tccttgtcga gttcgcggac ggcctcggga tgaccgactg
     8401 caatggcgag gaggtctgcc gagagctggc tgacgctggt gccgcgttgg gcggccattt
     8461 cgcgcagacg cgcgtacacg actgcggcag cgcggacctt gatctgctcg cgctgcccct
     8521 tatgcggttg ggccatgccg acgatcgtcc cccagagaaa gtcctgtacc tggtaccgac
     8581 acgccgaagc ggtgctcggt tttcggccga gttcaccccg gcatgactgc cggcgagaca
     8641 aagaagtcaa cgccaggcac tgcgcgaaca ctttccatct gaaggcgttg tctgacttgg
     8701 gagttggggg cgttcggcgc gtgattttgt cggtggtccc caatacgctg gcccaatgga
     8761 gcacccctac ccgacgggac caactaccca tccggctccc aactgcacac tcccccgctc
     8821 tcccgtcgag ccctggtggt ggaacaccag cgacagccga gcacccccaa ccacctgtac
     8881 caaccaggag gaacacatgc gtcgtttcga ggacgtttcc gggccgctga gagccgctgt
     8941 ggcggccgta cacgccgcct tagacccgtt agaccccctg ccgcctgaat gcgcgggtac
     9001 gagccacaca gcgcccgaac ttacggagct ggtgggctca cgcgttgcgc tcggtcgttc
     9061 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     9121 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     9181 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     9241 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     9301 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     9361 cctttctccc ctcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     9421 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     9481 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     9541 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     9601 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     9661 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     9721 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     9781 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcatgga actagtggtc
     9841 tgacgctcat ggaacaataa acgaaaggct cagtcgaaag actgggcctt tcgtttatct
     9901 gttgtttgaa aaaaaaaagc cccgcgattg cggggctttt ttttttcata tgtctgacca
     9961 gggaaaatag ccctctgacc tggggatttg act
//
