LOCUS       sequence-308739-        8881 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Sth1Cas9, TetR, KanR, L5Int, attP to create frameshifts and gene 
            deletion in Mycobacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8881)
  AUTHORS   Meijers AS, Troost R, Ummels R, Maaskant J, Speer A, Nejentsev S, 
            Bitter W, Kuijl CP
  TITLE     Efficient genome editing in pathogenic mycobacteria using 
            Streptococcus thermophilus CRISPR1-Cas9.
  JOURNAL   Tuberculosis (Edinb). 2020 Sep;124:101983. doi: 
            10.1016/j.tube.2020.101983. Epub 2020 Aug 12.
  PUBMED    32829077
REFERENCE   2  (bases 1 to 8881)
  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: 
            "Tuberculosis (Edinb)."; date: "2020-09"; volume: "124:101983. doi";
            pages: " 10.1016/j.tube.2020.101983. Epub 2020 Aug 12"
COMMENT     Sequence Label: pCRISPRx-Sth1Cas9-L5
FEATURES             Location/Qualifiers
     source          1..8881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(499..1087)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(579..598)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1270..1288
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1300..1318
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      4798..4825
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             5215..5838
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(5288..5307)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(7841..7860)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             7852..8667
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(7925..7944)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 atcaataaac gaaaggctca gtcgaaagac tgggcctttc gtttatctgt tgtttgaaaa
       61 aaaaaagcgc cgcaactgcg gcgctttttt ttttgaattc tctgaccagg gaaaatagcc
      121 ctctgacctg gggatttgcg atccctatca gtgatagata taatctggga ggagacgatt
      181 aatgcgtctc ggtttttgta ctcgaaagaa gctacaaaga taaggcttca tgccgaaatc
      241 aacaccctgt cattttatgg cagggtgttt tttttttgtc gacttgggga ccctagaggt
      301 cccctttttt atttttttgg atgaagagca ttatcaatgg ctcttcacag gaagcttaaa
      361 aaaaaaagcc ccgcgattgc ggggcttttt ttttttaata cggttatcca cagaatcagg
      421 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
      481 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
      541 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      601 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      661 ctttctcccc tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
      721 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
      781 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      841 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
      901 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
      961 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1021 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     1081 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcatggaa caataaacga
     1141 aaggctcagt cgaaagactg ggcctttcgt ttatctgttg tttgaaaaaa aaaagccccg
     1201 cgattgcggg gctttttttt ttcatatgtc tgaccaggga aaatagccct ctgacctggg
     1261 gatttgactt ccctatcagt gatagagata atctgggagt ccctatcagt gatagagaag
     1321 gcggtatcga tatgtcggac ctggtgctgg gcctggacat cggcatcggc tcggtgggcg
     1381 tgggcatcct gaacaaggtg accggcgaga tcatccacaa gaactcgcgc atcttcccgg
     1441 ccgcccaggc cgagaacaac ctggtgcgcc gcaccaaccg ccagggccgc cgcctggccc
     1501 gccgcaagaa gcaccgccgc gtgcgcctga accgcctgtt cgaggagtcg ggcctgatca
     1561 ccgacttcac caagatctcg atcaacctga acccgtacca gctgcgcgtg aagggcctga
     1621 ccgacgagct gtcgaacgag gagctgttca tcgccctgaa gaacatggtg aagcaccgcg
     1681 gcatctcgta cctggacgac gcctcggacg acggcaactc gtcggtgggc gactacgccc
     1741 agatcgtgaa ggagaactcg aagcagctgg agaccaagac cccgggccag atccagctgg
     1801 agcgctacca gacctacggc cagctgcgcg gcgacttcac cgtggagaag gacggcaaga
     1861 agcaccgcct gatcaacgtg ttcccgacct cggcctaccg ctcggaggcc ctgcgcatcc
     1921 tgcagaccca gcaggagttc aacccgcaga tcaccgacga gttcatcaac cgctacctgg
     1981 agatcctgac cggcaagcgc aagtactacc acggcccggg caacgagaag tcgcgcaccg
     2041 actacggccg ctaccgcacc tcgggcgaga ccctggacaa catcttcggc atcctgatcg
     2101 gcaagtgcac cttctacccg gacgagttcc gcgccgccaa ggcctcgtac accgcccagg
     2161 agttcaacct gctgaacgac ctgaacaacc tgaccgtgcc gaccgagacc aagaagctgt
     2221 cgaaggagca gaagaaccag atcatcaact acgtgaagaa cgagaaggcc atgggcccgg
     2281 ccaagctgtt caagtacatc gccaagctgc tgtcgtgcga cgtggccgac atcaagggct
     2341 accgcatcga caagtcgggc aaggccgaga tccacacctt cgaggcctac cgcaagatga
     2401 agaccctgga gaccctggac atcgagcaga tggaccgcga gaccctggac aagctggcct
     2461 acgtgctgac cctgaacacc gagcgcgagg gcatccagga ggccctggag cacgagttcg
     2521 ccgacggctc gttctcgcag aagcaggtgg acgagctggt gcagttccgc aaggccaact
     2581 cgtcgatctt cggcaagggc tggcacaact tctcggtgaa gctgatgatg gagctgatcc
     2641 cggagctgta cgagacctcg gaggagcaga tgaccatcct gacccgcctg ggcaagcaga
     2701 agaccacctc gtcgtcgaac aagaccaagt acatcgacga gaagctgctg accgaggaga
     2761 tctacaaccc ggtggtggcc aagtcggtgc gccaggccat caagatcgtg aacgccgcca
     2821 tcaaggagta cggcgacttc gacaacatcg tgatcgagat ggcccgcgag accaacgagg
     2881 acgacgagaa gaaggccatc cagaagatcc agaaggccaa caaggacgag aaggacgccg
     2941 ccatgctgaa ggccgccaac cagtacaacg gcaaggccga gctgccgcac tcggtgttcc
     3001 acggccacaa gcagctggcc accaagatcc gcctgtggca ccagcagggc gagcgctgcc
     3061 tgtacaccgg caagaccatc tcgatccacg acctgatcaa caactcgaac cagttcgagg
     3121 tggaccacat cctgccgctg tcgatcacct tcgacgactc gctggccaac aaggtgctgg
     3181 tgtacgccac cgccaaccag gagaagggcc agcgcacccc gtaccaggcc ctggactcga
     3241 tggacgacgc ctggtcgttc cgcgagctga aggccttcgt gcgcgagtcg aagaccctgt
     3301 cgaacaagaa gaaggagtac ctgctgaccg aggaggacat ctcgaagttc gacgtgcgca
     3361 agaagttcat cgagcgcaac ctggtggaca cccgctacgc ctcgcgcgtg gtgctgaacg
     3421 ccctgcagga gcacttccgc gcccacaaga tcgacaccaa ggtgtcggtg gtgcgcggcc
     3481 agttcacctc gcagctgcgc cgccactggg gcatcgagaa gacccgcgac acctaccacc
     3541 accacgccgt ggacgccctg atcatcgccg cctcgtcgca gctgaacctg tggaagaagc
     3601 agaagaacac cctggtgtcg tactcggagg accagctgct ggacatcgag accggcgagc
     3661 tgatctcgga cgacgagtac aaggagtcgg tgttcaaggc cccgtaccag cacttcgtgg
     3721 acaccctgaa gtcgaaggag ttcgaggact cgatcctgtt ctcgtaccag gtggactcga
     3781 agttcaaccg caagatctcg gacgccacca tctacgccac ccgccaggcc aaggtgggca
     3841 aggacaaggc cgacgagacc tacgtgctgg gcaagatcaa ggacatctac acccaggacg
     3901 gctacgacgc cttcatgaag atctacaaga aggacaagtc gaagttcctg atgtaccgcc
     3961 acgacccgca gaccttcgag aaggtgatcg agccgatcct ggagaactac ccgaacaagc
     4021 agatcaacga caagggcaag gaggtgccgt gcaacccgtt cctgaagtac aaggaggagc
     4081 acggctacat ccgcaagtac tcgaagaagg gcaacggccc ggagatcaag tcgctgaagt
     4141 actacgactc gaagctgggc aaccacatcg acatcacccc gaaggactcg aacaacaagg
     4201 tggtgctgca gtcggtgtcg ccgtggcgcg ccgacgtgta cttcaacaag accaccggca
     4261 agtacgagat cctgggcctg aagtacgccg acctgcagtt cgacaagggc accggcacct
     4321 acaagatctc gcaggagaag tacaacgaca tcaagaagaa ggagggcgtg gactcggact
     4381 cggagttcaa gttcaccctg tacaagaacg acctgctgct ggtgaaggac accgagacca
     4441 aggagcagca gctgttccgc ttcctgtcgc gcaccatgcc gaagcagaag cactacgtgg
     4501 agctgaagcc gtacgacaag cagaagttcg agggcggcga ggccctgatc aaggtgctgg
     4561 gcaacgtggc caactcgggc cagtgcaaga agggcctggg caagtcgaac atctcgatct
     4621 acaaggtgcg caccgacgtg ctgggcaacc agcacatcat caagaacgag ggcgacaagc
     4681 cgaagctgga cttctaaata ctctggccat ttttctttac cgcctttgag tgagctgata
     4741 ccgctcgccg cagccgaacg accgagcgca acgcgtgcgg ccgctgatta gctaagcaga
     4801 aggccatcct gacggatggc ctttttctag aaaaaaaaaa agcgccgcaa ctgcggcgct
     4861 tttttttttt taattaaaga aatattggat cgtcggcacc gtcacggccg tgggaggcgg
     4921 cacgatccgc gacgtgatga tcggccgcat ccccacggtg ctgcgcagtg agctctacgc
     4981 catcccggcg ttgatctgtg cgttcgcacg cacaggcccg gtgtgagaag ggtctctgac
     5041 gagcgggaga acccacccgg ggtgggcgag tttgtcctgc gtgtgctcgg tcgagtaggc
     5101 tctgggagta cccgtgtgta cgaccagcac ggcatacatc atttcgacgc cgagagattc
     5161 gccgcccgaa atgagcacga tccgcatggt accatcagga ggaatcactt cgcaatgtct
     5221 agattagata aaagtaaagt gattaacagc gcattagagc tgcttaatga ggtcggaatc
     5281 gaaggtttaa caacccgtaa actcgcccag aagcttggtg tagagcagcc tacattgtat
     5341 tggcatgtaa aaaataagcg ggccctgctc gacgccttag ccattgagat gttagatagg
     5401 caccatactc acttttgccc tttagaaggg gaaagctggc aagatttttt acgtaataac
     5461 gctaaaagtt ttagatgtgc tttactaagt catcgcgatg gagcaaaagt acatttaggt
     5521 acacggccta cagaaaaaca gtatgaaact ctcgaaaatc aattagcctt tttatgccaa
     5581 caaggttttt cactagagaa cgcgttatat gcactcagcg ctgtggggca ttttacctta
     5641 ggttgcgtat tggaagatca agagcatcaa gtcgctaaag aagaaaggga aacacctact
     5701 actgatagta tgccgccatt attacgacaa gctatcgaat tatttgatca ccaaggtgca
     5761 gagccagcct tcttattcgg ccttgaattg atcatctgcg gattagaaaa acaacttaaa
     5821 tgtgaaagtg ggtcttaatg accatgggct agcgctttag ctaattaatt taaaataaaa
     5881 aaggggacct ctagggtccc caattaaata gttaacacta gtattaaaaa aaaaagcccc
     5941 gcgattgcgg ggcttttttt tttcgacggc gagggttccg accgctgcaa ctcccggtgc
     6001 aaccttgtcc cggtctattc tcttcactgc accagctcca atctggtgtg aatgcccctc
     6061 gtctgttcgc gcaggcgggg ggctctattc gtttgtcagc atcgaaagta gccagatcag
     6121 ggatgcgttg caaccgcgta tgcccaggtc agaagagtcg cacaagagtt gcagacccct
     6181 ggaaagaaaa atggccagag ggcgaaaaca ccctctgacc agcggagcgg gcgacgggaa
     6241 tcgaacccgc gtagctagtt tggaagaatg ggtgtctgcc gaccacatat atgggccggt
     6301 caagataggt ttttaccccc tctcggctgc atcctctaag tggaaagaaa ttgcaggtcg
     6361 tagaagcgcg ttgaagcctg agagttgcac aggagttgca acccggtagc cttgttcacg
     6421 acgagaggag acctagttgg cacgtcgcgg atggggatcg ctgaagactc agcgcagcgg
     6481 gaggatccaa gcctcatacg tcaacccgca ggacggtgtg aggtactacg cgctgcagac
     6541 ctacgacaac aagatggacg ccgaagcctg gctcgcgggc gagaagcggc tcatcgagat
     6601 ggagacctgg acccctccac aggaccgggc gaagaaggca gccgccagcg ccatcacgct
     6661 ggaggagtac acccggaagt ggctcgtgga gcgcgacctc gcagacggca ccagggatct
     6721 gtacagcggg cacgcggagc gccgcatcta cccggtgcta ggtgaagtgg cggtcacaga
     6781 gatgacgcca gctctggtgc gtgcgtggtg ggccgggatg ggtaggaagc acccgactgc
     6841 ccgccggcat gcctacaacg tcctccgggc ggtgatgaac acagcggtcg aggacaagct
     6901 gatcgcagag aacccgtgcc ggatcgagca gaaggcagcc gatgagcgcg acgtagaggc
     6961 gctgacgcct gaggagctgg acatcgtcgc cgctgagatc ttcgtgcact accggatcgc
     7021 ggcatacatc ctggcgtgga cgagcctccg gttcggagag ctgatcgagc ttcgccgcaa
     7081 ggacatcgtg gacgacggca tgacgatgaa gctccgggtg cgccgtggcg cttcccgcgt
     7141 ggggaacaag atcgtcgttg gcaacgccaa gaccgtccgg tcgaagcgtc ctgtgacggt
     7201 tccgcctcac gtcgcggaga tgatccgagc gcacatgaag gaccgtacga agatgaacaa
     7261 gggccccgag gcattcctgg tgaccacgac gcagggcaac cggctgtcga agtccgcgtt
     7321 caccaagtcg ctgaagcgtg gctacgccaa gatcggtcgg ccggaactcc gcatccacga
     7381 cctccgcgct gtcggcgcta cgttcgccgc tcaggcaggt gcgacgacca aggagctgat
     7441 ggcccgcctc ggtcacacga ctcctaggat ggcgatgaag taccagatgg cgtctgaggc
     7501 ccgcgacgag gctatcgctg aggcgatgtc caagctggcc aagacctcct gaaacgcaaa
     7561 aagcccccct cccaaggaca ctgagtccta aagagggggg tttcttgtca gtacgcgaag
     7621 aaccacgcct ggccgcgagc gccagcaccg ccgctctgtg cggagacctg ggcaccagcc
     7681 ccgccgccgc caggagcatt gccgttcccg ccagctagcc acctgacgtc gggggggggg
     7741 gaaagccacg ttgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
     7801 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     7861 attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta
     7921 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg
     7981 tatgggaagc cccatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     8041 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     8101 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
     8161 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     8221 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     8281 gatcgcgtat ttcgcctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     8341 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     8401 aatcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     8461 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     8521 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     8581 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     8641 catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag
     8701 agcattacgc tgacttgacg ggacggcggc tttgttgaat aaatcgaact tttgctgagt
     8761 tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca aagcaaaagt
     8821 tcaaaatcac caactggtcc acctacaaca aagctctcac caaccgtggc tccctcacga
     8881 t
//
