LOCUS       sequence-335569-        7670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Optimized M. tuberculosis CRISPRi plasmid; contains an L5 attP 
            region (no L5 Integrase) that allows for integration when 
            co-transformed with an L5 Integrase delivery plasmid (e.g. plRL19, 
            ID #163634).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7670)
  AUTHORS   Bosch B, DeJesus MA, Poulton NC, Zhang W, Engelhart CA, Zaveri A, 
            Lavalette S, Ruecker N, Trujillo C, Wallach JB, Li S, Ehrt S, Chait 
            BT, Schnappinger D, Rock JM
  TITLE     Genome-wide gene expression tuning reveals diverse vulnerabilities 
            of M. tuberculosis.
  JOURNAL   Cell. 2021 Jul 16. pii: S0092-8674(21)00824-2. doi: 
            10.1016/j.cell.2021.06.033.
  PUBMED    34297925
REFERENCE   2  (bases 1 to 7670)
  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: "Cell. 2021 
            Jul 16. pii: S0092-8674(21)00824-2. doi: 
            10.1016/j.cell.2021.06.033."
COMMENT     Sequence Label: plRL58
FEATURES             Location/Qualifiers
     source          1..7670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2428..2455
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4260..4279)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4271..5086
                     /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(4344..4363)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5799..6387)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5879..5898)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6570..6588
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6600..6618
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 tccacacctt cgaggcctac cgcaagatga agaccctgga gaccctggac atcgagcaga
       61 tggaccgcga gaccctggac aagctggcct acgtgctgac cctgaacacc gagcgcgagg
      121 gcatccagga ggccctggag cacgagttcg ccgacggctc gttctcgcag aagcaggtgg
      181 acgagctggt gcagttccgc aaggccaact cgtcgatctt cggcaagggc tggcacaact
      241 tctcggtgaa gctgatgatg gagctgatcc cggagctgta cgagacctcg gaggagcaga
      301 tgaccatcct gacccgcctg ggcaagcaga agaccacctc gtcgtcgaac aagaccaagt
      361 acatcgacga gaagctgctg accgaggaga tctacaaccc ggtggtggcc aagtcggtgc
      421 gccaggccat caagatcgtg aacgccgcca tcaaggagta cggcgacttc gacaacatcg
      481 tgatcgagat ggcccgcgag accaacgagg acgacgagaa gaaggccatc cagaagatcc
      541 agaaggccaa caaggacgag aaggacgccg ccatgctgaa ggccgccaac cagtacaacg
      601 gcaaggccga gctgccgcac tcggtgttcc acggccacaa gcagctggcc accaagatcc
      661 gcctgtggca ccagcagggc gagcgctgcc tgtacaccgg caagaccatc tcgatccacg
      721 acctgatcaa caactcgaac cagttcgagg tggacgccat cctgccgctg tcgatcacct
      781 tcgacgactc gctggccaac aaggtgctgg tgtacgccac cgccaaccag gagaagggcc
      841 agcgcacccc gtaccaggcc ctggactcga tggacgacgc ctggtcgttc cgcgagctga
      901 aggccttcgt gcgcgagtcg aagaccctgt cgaacaagaa gaaggagtac ctgctgaccg
      961 aggaggacat ctcgaagttc gacgtgcgca agaagttcat cgagcgcaac ctggtggaca
     1021 cccgctacgc ctcgcgcgtg gtgctgaacg ccctgcagga gcacttccgc gcccacaaga
     1081 tcgacaccaa ggtgtcggtg gtgcgcggcc agttcacctc gcagctgcgc cgccactggg
     1141 gcatcgagaa gacccgcgac acctaccacc accacgccgt ggacgccctg atcatcgccg
     1201 cctcgtcgca gctgaacctg tggaagaagc agaagaacac cctggtgtcg tactcggagg
     1261 accagctgct ggacatcgag accggcgagc tgatctcgga cgacgagtac aaggagtcgg
     1321 tgttcaaggc cccgtaccag cacttcgtgg acaccctgaa gtcgaaggag ttcgaggact
     1381 cgatcctgtt ctcgtaccag gtggactcga agttcaaccg caagatctcg gacgccacca
     1441 tctacgccac ccgccaggcc aaggtgggca aggacaaggc cgacgagacc tacgtgctgg
     1501 gcaagatcaa ggacatctac acccaggacg gctacgacgc cttcatgaag atctacaaga
     1561 aggacaagtc gaagttcctg atgtaccgcc acgacccgca gaccttcgag aaggtgatcg
     1621 agccgatcct ggagaactac ccgaacaagc agatcaacga caagggcaag gaggtgccgt
     1681 gcaacccgtt cctgaagtac aaggaggagc acggctacat ccgcaagtac tcgaagaagg
     1741 gcaacggccc ggagatcaag tcgctgaagt actacgactc gaagctgggc aaccacatcg
     1801 acatcacccc gaaggactcg aacaacaagg tggtgctgca gtcggtgtcg ccgtggcgcg
     1861 ccgacgtgta cttcaacaag accaccggca agtacgagat cctgggcctg aagtacgccg
     1921 acctgcagtt cgacaagggc accggcacct acaagatctc gcaggagaag tacaacgaca
     1981 tcaagaagaa ggagggcgtg gactcggact cggagttcaa gttcaccctg tacaagaacg
     2041 acctgctgct ggtgaaggac accgagacca aggagcagca gctgttccgc ttcctgtcgc
     2101 gcaccatgcc gaagcagaag cactacgtgg agctgaagcc gtacgacaag cagaagttcg
     2161 agggcggcga ggccctgatc aaggtgctgg gcaacgtggc caactcgggc cagtgcaaga
     2221 agggcctggg caagtcgaac atctcgatct acaaggtgcg caccgacgtg ctgggcaacc
     2281 agcacatcat caagaacgag ggcgacaagc cgaagctgga cttctaaata ctctggccat
     2341 ttttctttac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2401 acgcgtgcgg ccgctgatta gctaagcaga aggccatcct gacggatggc ctttttctag
     2461 aaaaaaaaaa agcgccgcaa ctgcggcgct tttttttttt taattaagtg atagattgcg
     2521 atgcgtgtgc tcggttgata taatctgtca gatcaggagg acttcgcatg tcgcggttgg
     2581 ataagtccaa ggtcatcaac tcggcgctgg agctgctgaa cgaggtcggt atcgagggac
     2641 tgaccacgcg caagttggcc caaaagctcg gcgtggaaca accgaccctc tactggcacg
     2701 tgaagaacaa gcgcgcgctg ctggatgcgt tggccatcga gatgctggat cgccatcaca
     2761 cgcacttttg ccccctggag ggcgagagct ggcaggattt tctgcggaac aacgcgaagt
     2821 cgttccggtg cgccctcctg agccatcggg atggcgccaa agtccacttg ggcacccgcc
     2881 cgaccgagaa gcaatacgag accctggaga accagctggc cttcctctgt caacagggtt
     2941 tttcgttgga gaacgccctg tatgccctgt ccgccgtggg acacttcacc ttgggctgcg
     3001 tgttggagga tcaggaacat caggtggcga aagaggaacg ggagaccccc acgacggatt
     3061 cgatgccccc gttgttgcgc caggcgatcg aactgtttga ccaccagggc gccgaacccg
     3121 cctttttgtt cgggctggag ctgatcatct gcgggctgga aaagcaactg aagtgcgaaa
     3181 gcggttcgtg ataacgccga aaaccccgct tcggcggggt tttgccgcgg tctgcgttgt
     3241 cgggaagatt taaaataaaa aaggggacct ctagggtccc caattaaata gttaacacta
     3301 gtattaaaaa aaaaagcccc gcgattgcgg ggcttttttt tttcgacggc gagggttccg
     3361 accgctgcaa ctcccggtgc aaccttgtcc cggtctattc tcttcactgc accagctcca
     3421 atctggtgtg aatgcccctc gtctgttcgc gcaggcgggg ggctctattc gtttgtcagc
     3481 atcgaaagta gccagatcag ggatgcgttg caaccgcgta tgcccaggtc agaagagtcg
     3541 cacaagagtt gcagacccct ggaaagaaaa atggccagag ggcgaaaaca ccctctgacc
     3601 agcggagcgg gcgacgggaa tcgaacccgc gtagctagtt tggaagaatg ggtgtctgcc
     3661 gaccacatat atgggccggt caagataggt ttttaccccc tctcggctgc atcctctaag
     3721 tggaaagaaa ttgcaggtcg tagaagcgcg ttgaagcctg agagttgcac aggagttgca
     3781 acccggtagc cttgttcacg acgagaggag acctagttgg cacgtcgcgg atggggatcg
     3841 ctgaagactc agcgcagcgg gaggatccac ttgcactgca ccctaggatg gcgatgaagt
     3901 accagatggc gtctgaggcc cgcgacgagg ctatcgctga ggcgatgtcc aagctggcca
     3961 agacctcctg aaacgcaaaa agcccccctc ccaaggacac tgagtcctaa agaggggggt
     4021 ttcttgtcag tacgcgaaga accacgcctg gccgcgagcg ccagcaccgc cgctctgtgc
     4081 ggagacctgg gcaccagccc cgccgccgcc aggagcattg ccgttcccgc cagctagcca
     4141 cctgacgtcg gggggggggg aaagccacgt tgtgtctcaa aatctctgat gttacattgc
     4201 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
     4261 aaggggtgtt atgagccata ttcaacggga aacgtcttgc tcgaggccgc gattaaattc
     4321 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     4381 tgcgacaatc tatcgcttgt atgggaagcc ccatgcgcca gagttgtttc tgaaacatgg
     4441 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     4501 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     4561 caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     4621 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     4681 taattgtcct tttaacagcg atcgcgtatt tcgcctcgct caggcgcaat cacgaatgaa
     4741 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     4801 agtctggaaa gaaatgcata atcttttgcc attctcaccg gattcagtcg tcactcatgg
     4861 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     4921 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     4981 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     5041 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     5101 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcggct ttgttgaata
     5161 aatcgaactt ttgctgagtt gaaggatcag atcacgcatc ttcccgacaa cgcagaccgt
     5221 tccgtggcaa agcaaaagtt caaaatcacc aactggtcca cctacaacaa agctctcacc
     5281 aaccgtggct ccctcacgat atcaataaac gaaaggctca gtcgaaagac tgggcctttc
     5341 gtttatctgt tgtttgaaaa aaaaaagcgc cgcaactgcg gcgctttttt ttttgaattc
     5401 tctgaccagg gaaaatagcc ctctgacctg gggatttgcg atccctatca gtgatagata
     5461 taatctggga ggagacgatt aatgcgtctc ggtttttgta ctcgaaagaa gctacaaaga
     5521 taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt tttttttgtc
     5581 gacttgggga ccctagaggt cccctttttt atttttttgg atgaagagca ttatcaatgg
     5641 ctcttcacag gaagcttaaa aaaaaaagcc ccgcgattgc ggggcttttt ttttttaata
     5701 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5761 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5821 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5881 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5941 cttaccggat acctgtccgc ctttctcccc tcgggaagcg tggcgctttc tcatagctca
     6001 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6061 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6121 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6181 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     6241 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6301 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6361 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6421 gctcatggaa caataaacga aaggctcagt cgaaagactg ggcctttcgt ttatctgttg
     6481 tttgaaaaaa aaaagccccg cgattgcggg gctttttttt ttcatatgtc tgaccaggga
     6541 aaatagccct ctgacctggg gatttgactt ccctatcagt gatagagata atctgggagt
     6601 ccctatcagt gatagagaac gcggtatcga tatgtcggac ctggtgctgg gcctggccat
     6661 cggcatcggc tcggtgggcg tgggcatcct gaacaaggtg accggcgaga tcatccacaa
     6721 gaactcgcgc atcttcccgg ccgcccaggc cgagaacaac ctggtgcgcc gcaccaaccg
     6781 ccagggccgc cgcctggccc gccgcaagaa gcaccgccgc gtgcgcctga accgcctgtt
     6841 cgaggagtcg ggcctgatca ccgacttcac caagatctcg atcaacctga acccgtacca
     6901 gctgcgcgtg aagggcctga ccgacgagct gtcgaacgag gagctgttca tcgccctgaa
     6961 gaacatggtg aagcaccgcg gcatctcgta cctggacgac gcctcggacg acggcaactc
     7021 gtcggtgggc gactacgccc agatcgtgaa ggagaactcg aagcagctgg agaccaagac
     7081 cccgggccag atccagctgg agcgctacca gacctacggc cagctgcgcg gcgacttcac
     7141 cgtggagaag gacggcaaga agcaccgcct gatcaacgtg ttcccgacct cggcctaccg
     7201 ctcggaggcc ctgcgcatcc tgcagaccca gcaggagttc aacccgcaga tcaccgacga
     7261 gttcatcaac cgctacctgg agatcctgac cggcaagcgc aagtactacc acggcccggg
     7321 caacgagaag tcgcgcaccg actacggccg ctaccgcacc tcgggcgaga ccctggacaa
     7381 catcttcggc atcctgatcg gcaagtgcac cttctacccg gacgagttcc gcgccgccaa
     7441 ggcctcgtac accgcccagg agttcaacct gctgaacgac ctgaacaacc tgaccgtgcc
     7501 gaccgagacc aagaagctgt cgaaggagca gaagaaccag atcatcaact acgtgaagaa
     7561 cgagaaggcc atgggcccgg ccaagctgtt caagtacatc gccaagctgc tgtcgtgcga
     7621 cgtggccgac atcaagggct accgcatcga caagtcgggc aaggccgaga
//
