LOCUS       sequence-335571-        7670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Optimized M. smegmatis 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: plRL61
FEATURES             Location/Qualifiers
     source          1..7670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    581..599
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    611..629
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      4109..4136
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(5941..5960)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5952..6767
                     /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(6025..6044)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(7480..7670,1..398))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7560..7579)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
       61 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
      121 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
      181 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
      241 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
      301 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
      361 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
      421 cggggtctga cgctcatgga acaataaacg aaaggctcag tcgaaagact gggcctttcg
      481 tttatctgtt gtttgaaaaa aaaaagcccc gcgattgcgg ggcttttttt tttcatatgt
      541 ctgaccaggg aaaatagccc tctgacctgg ggatttgact tccctatcag tgatagagat
      601 aatctgggag tccctatcag tgatagagaa ggcgctatcg atatgtcgga cctggtgctg
      661 ggcctggcca tcggcatcgg ctcggtgggc gtgggcatcc tgaacaaggt gaccggcgag
      721 atcatccaca agaactcgcg catcttcccg gccgcccagg ccgagaacaa cctggtgcgc
      781 cgcaccaacc gccagggccg ccgcctggcc cgccgcaaga agcaccgccg cgtgcgcctg
      841 aaccgcctgt tcgaggagtc gggcctgatc accgacttca ccaagatctc gatcaacctg
      901 aacccgtacc agctgcgcgt gaagggcctg accgacgagc tgtcgaacga ggagctgttc
      961 atcgccctga agaacatggt gaagcaccgc ggcatctcgt acctggacga cgcctcggac
     1021 gacggcaact cgtcggtggg cgactacgcc cagatcgtga aggagaactc gaagcagctg
     1081 gagaccaaga ccccgggcca gatccagctg gagcgctacc agacctacgg ccagctgcgc
     1141 ggcgacttca ccgtggagaa ggacggcaag aagcaccgcc tgatcaacgt gttcccgacc
     1201 tcggcctacc gctcggaggc cctgcgcatc ctgcagaccc agcaggagtt caacccgcag
     1261 atcaccgacg agttcatcaa ccgctacctg gagatcctga ccggcaagcg caagtactac
     1321 cacggcccgg gcaacgagaa gtcgcgcacc gactacggcc gctaccgcac ctcgggcgag
     1381 accctggaca acatcttcgg catcctgatc ggcaagtgca ccttctaccc ggacgagttc
     1441 cgcgccgcca aggcctcgta caccgcccag gagttcaacc tgctgaacga cctgaacaac
     1501 ctgaccgtgc cgaccgagac caagaagctg tcgaaggagc agaagaacca gatcatcaac
     1561 tacgtgaaga acgagaaggc catgggcccg gccaagctgt tcaagtacat cgccaagctg
     1621 ctgtcgtgcg acgtggccga catcaagggc taccgcatcg acaagtcggg caaggccgag
     1681 atccacacct tcgaggccta ccgcaagatg aagaccctgg agaccctgga catcgagcag
     1741 atggaccgcg agaccctgga caagctggcc tacgtgctga ccctgaacac cgagcgcgag
     1801 ggcatccagg aggccctgga gcacgagttc gccgacggct cgttctcgca gaagcaggtg
     1861 gacgagctgg tgcagttccg caaggccaac tcgtcgatct tcggcaaggg ctggcacaac
     1921 ttctcggtga agctgatgat ggagctgatc ccggagctgt acgagacctc ggaggagcag
     1981 atgaccatcc tgacccgcct gggcaagcag aagaccacct cgtcgtcgaa caagaccaag
     2041 tacatcgacg agaagctgct gaccgaggag atctacaacc cggtggtggc caagtcggtg
     2101 cgccaggcca tcaagatcgt gaacgccgcc atcaaggagt acggcgactt cgacaacatc
     2161 gtgatcgaga tggcccgcga gaccaacgag gacgacgaga agaaggccat ccagaagatc
     2221 cagaaggcca acaaggacga gaaggacgcc gccatgctga aggccgccaa ccagtacaac
     2281 ggcaaggccg agctgccgca ctcggtgttc cacggccaca agcagctggc caccaagatc
     2341 cgcctgtggc accagcaggg cgagcgctgc ctgtacaccg gcaagaccat ctcgatccac
     2401 gacctgatca acaactcgaa ccagttcgag gtggacgcca tcctgccgct gtcgatcacc
     2461 ttcgacgact cgctggccaa caaggtgctg gtgtacgcca ccgccaacca ggagaagggc
     2521 cagcgcaccc cgtaccaggc cctggactcg atggacgacg cctggtcgtt ccgcgagctg
     2581 aaggccttcg tgcgcgagtc gaagaccctg tcgaacaaga agaaggagta cctgctgacc
     2641 gaggaggaca tctcgaagtt cgacgtgcgc aagaagttca tcgagcgcaa cctggtggac
     2701 acccgctacg cctcgcgcgt ggtgctgaac gccctgcagg agcacttccg cgcccacaag
     2761 atcgacacca aggtgtcggt ggtgcgcggc cagttcacct cgcagctgcg ccgccactgg
     2821 ggcatcgaga agacccgcga cacctaccac caccacgccg tggacgccct gatcatcgcc
     2881 gcctcgtcgc agctgaacct gtggaagaag cagaagaaca ccctggtgtc gtactcggag
     2941 gaccagctgc tggacatcga gaccggcgag ctgatctcgg acgacgagta caaggagtcg
     3001 gtgttcaagg ccccgtacca gcacttcgtg gacaccctga agtcgaagga gttcgaggac
     3061 tcgatcctgt tctcgtacca ggtggactcg aagttcaacc gcaagatctc ggacgccacc
     3121 atctacgcca cccgccaggc caaggtgggc aaggacaagg ccgacgagac ctacgtgctg
     3181 ggcaagatca aggacatcta cacccaggac ggctacgacg ccttcatgaa gatctacaag
     3241 aaggacaagt cgaagttcct gatgtaccgc cacgacccgc agaccttcga gaaggtgatc
     3301 gagccgatcc tggagaacta cccgaacaag cagatcaacg acaagggcaa ggaggtgccg
     3361 tgcaacccgt tcctgaagta caaggaggag cacggctaca tccgcaagta ctcgaagaag
     3421 ggcaacggcc cggagatcaa gtcgctgaag tactacgact cgaagctggg caaccacatc
     3481 gacatcaccc cgaaggactc gaacaacaag gtggtgctgc agtcggtgtc gccgtggcgc
     3541 gccgacgtgt acttcaacaa gaccaccggc aagtacgaga tcctgggcct gaagtacgcc
     3601 gacctgcagt tcgacaaggg caccggcacc tacaagatct cgcaggagaa gtacaacgac
     3661 atcaagaaga aggagggcgt ggactcggac tcggagttca agttcaccct gtacaagaac
     3721 gacctgctgc tggtgaagga caccgagacc aaggagcagc agctgttccg cttcctgtcg
     3781 cgcaccatgc cgaagcagaa gcactacgtg gagctgaagc cgtacgacaa gcagaagttc
     3841 gagggcggcg aggccctgat caaggtgctg ggcaacgtgg ccaactcggg ccagtgcaag
     3901 aagggcctgg gcaagtcgaa catctcgatc tacaaggtgc gcaccgacgt gctgggcaac
     3961 cagcacatca tcaagaacga gggcgacaag ccgaagctgg acttctaaat actctggcca
     4021 tttttcttta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4081 aacgcgtgcg gccgctgatt agctaagcag aaggccatcc tgacggatgg cctttttcta
     4141 gaaaaaaaaa aagcgccgca actgcggcgc tttttttttt ttaattaagt gatagattgc
     4201 gatgcgtgtg ctcggttgat ataatctgtc agatcaggag gacttcgcat gtcgcggttg
     4261 gataagtcca aggtcatcaa ctcggcgctg gagctgctga acgaggtcgg tatcgaggga
     4321 ctgaccacgc gcaagttggc ccaaaagctc ggcgtggaac aaccgaccct ctactggcac
     4381 gtgaagaaca agcgcgcgct gctggatgcg ttggccatcg agatgctgga tcgccatcac
     4441 acgcactttt gccccctgga gggcgagagc tggcaggatt ttctgcggaa caacgcgaag
     4501 tcgttccggt gcgccctcct gagccatcgg gatggcgcca aagtccactt gggcacccgc
     4561 ccgaccgaga agcaatacga gaccctggag aaccagctgg ccttcctctg tcaacagggt
     4621 ttttcgttgg agaacgccct gtatgccctg tccgccgtgg gacacttcac cttgggctgc
     4681 gtgttggagg atcaggaaca tcaggtggcg aaagaggaac gggagacccc cacgacggat
     4741 tcgatgcccc cgttgttgcg ccaggcgatc gaactgtttg accaccaggg cgccgaaccc
     4801 gcctttttgt tcgggctgga gctgatcatc tgcgggctgg aaaagcaact gaagtgcgaa
     4861 agcggttcgt gataacgccg aaaaccccgc ttcggcgggg ttttgccgcg gtctgcgttg
     4921 tcgggaagat ttaaaataaa aaaggggacc tctagggtcc ccaattaaat agttaacact
     4981 agtattaaaa aaaaaagccc cgcgattgcg gggctttttt ttttcgacgg cgagggttcc
     5041 gaccgctgca actcccggtg caaccttgtc ccggtctatt ctcttcactg caccagctcc
     5101 aatctggtgt gaatgcccct cgtctgttcg cgcaggcggg gggctctatt cgtttgtcag
     5161 catcgaaagt agccagatca gggatgcgtt gcaaccgcgt atgcccaggt cagaagagtc
     5221 gcacaagagt tgcagacccc tggaaagaaa aatggccaga gggcgaaaac accctctgac
     5281 cagcggagcg ggcgacggga atcgaacccg cgtagctagt ttggaagaat gggtgtctgc
     5341 cgaccacata tatgggccgg tcaagatagg tttttacccc ctctcggctg catcctctaa
     5401 gtggaaagaa attgcaggtc gtagaagcgc gttgaagcct gagagttgca caggagttgc
     5461 aacccggtag ccttgttcac gacgagagga gacctagttg gcacgtcgcg gatggggatc
     5521 gctgaagact cagcgcagcg ggaggatcca cttgcactgc accctaggat ggcgatgaag
     5581 taccagatgg cgtctgaggc ccgcgacgag gctatcgctg aggcgatgtc caagctggcc
     5641 aagacctcct gaaacgcaaa aagcccccct cccaaggaca ctgagtccta aagagggggg
     5701 tttcttgtca gtacgcgaag aaccacgcct ggccgcgagc gccagcaccg ccgctctgtg
     5761 cggagacctg ggcaccagcc ccgccgccgc caggagcatt gccgttcccg ccagctagcc
     5821 acctgacgtc gggggggggg gaaagccacg ttgtgtctca aaatctctga tgttacattg
     5881 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata
     5941 caaggggtgt tatgagccat attcaacggg aaacgtcttg ctcgaggccg cgattaaatt
     6001 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     6061 gtgcgacaat ctatcgcttg tatgggaagc cccatgcgcc agagttgttt ctgaaacatg
     6121 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     6181 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     6241 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag
     6301 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     6361 gtaattgtcc ttttaacagc gatcgcgtat ttcgcctcgc tcaggcgcaa tcacgaatga
     6421 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     6481 aagtctggaa agaaatgcat aatcttttgc cattctcacc ggattcagtc gtcactcatg
     6541 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     6601 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     6661 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     6721 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta
     6781 attggttgta acactggcag agcattacgc tgacttgacg ggacggcggc tttgttgaat
     6841 aaatcgaact tttgctgagt tgaaggatca gatcacgcat cttcccgaca acgcagaccg
     6901 ttccgtggca aagcaaaagt tcaaaatcac caactggtcc acctacaaca aagctctcac
     6961 caaccgtggc tccctcacga tatcaataaa cgaaaggctc agtcgaaaga ctgggccttt
     7021 cgtttatctg ttgtttgaaa aaaaaaagcg ccgcaactgc ggcgcttttt tttttgaatt
     7081 ctctgaccag ggaaaatagc cctctgacct ggggatttgc gatccctatc agtgatagat
     7141 ataatctggg aggagacgat taatgcgtct cggtttttgt actcgaaaga agctacaaag
     7201 ataaggcttc atgccgaaat caacaccctg tcattttatg gcagggtgtt ttttttttgt
     7261 cgacttgggg accctagagg tccccttttt tatttttttg gatgaagagc attatcaatg
     7321 gctcttcaca ggaagcttaa aaaaaaaagc cccgcgattg cggggctttt tttttttaat
     7381 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     7441 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     7501 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     7561 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     7621 gcttaccgga tacctgtccg cctttctccc ctcgggaagc gtggcgcttt
//
