LOCUS       sequence-468506-        4770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi-ART crRNA only plasmid encoding crystal violet-inducible 
            crRNA (for dRfxCas13d) with 2xBsaI spacer cloning site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4770)
  AUTHORS   Adler BA, Al-Shimary MJ, Patel JR, Armbruster EG, Colognori D, 
            Charles EJ, Miller KV, Lahiri A, Cui ML, Oromi-Bosch A, Voelker A, 
            Trinidad M, Lee J, Beurnier S, Boger R, Nomburg J, Barrangou R, 
            Mutalik VK, Schoeniger JS, Pogliano JA, Savage DF, Doudna JA, Cress 
            BF
  TITLE     CRISPRi-ART enables functional genomics of diverse bacteriophages 
            using RNA-binding dCas13d.
  JOURNAL   Nat Microbiol. 2025 Mar;10(3):694-709. doi: 
            10.1038/s41564-025-01935-7. Epub 2025 Feb 26.
  PUBMED    40011704
REFERENCE   2  (bases 1 to 4770)
  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"; doi: 
            "10.1038/s41564-025-01935-7"; journalName: "Nat Microbiol"; date: 
            "2025-03"; volume: "10"; issue: "3"; pages: "694-709"
COMMENT     Sequence Label: pBFC1171
FEATURES             Location/Qualifiers
     source          1..4770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             733..741
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     misc_RNA        1058..1087
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     CDS             1126..1155
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1171..1188
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1244..1261)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1414..1460
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2099..3049)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      3097..3319
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(3900..4691,4692..4760))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRLA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4523..4542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(4761..4770,1..95))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
       61 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
      121 gttagaacga ttgtctgatt cgttacctta cgaaaataac tcaagctgaa taacgtgctg
      181 caggtggcgg gtgaatgcgg catcgtctac ctccggcatt cccagcacgt aaatcccgtc
      241 cagtccacag accagcgaaa tcagccgcca ggcgatattt tcggcgctat cgcgcagggt
      301 aaattcgcca gcggcatggc ccgcgcgaat gatcctgacc gcttcgtcat gccacaggtt
      361 catggtcagc aggtacgcgc ttttgatttc cggatcgctg tcggccagca gctgcgcctg
      421 gcgccacagg cggatatagg gctccagccg tccgtcctcg ctgccgaggg cggaaaacag
      481 ctgttcacgc cagccggcgg tgcgcgaaag gcgctgcaga tccatcatct cgcggatcac
      541 gcggataaac gcctgggatt ttagctcgcc ggaggaggtg aaatggtggt ggacctggcc
      601 tgcggcgacg cccgcggcgg tggcgatgtt gcggaccgtc ataccggtaa acccttgatc
      661 cagcgcgacg cgcatggcgg cctgcatgat ggtttcccga cgttcttcgc gattcagata
      721 gcccatattg tcacctcctt atccacacat tatacgagcc ggatgattaa tggggaacct
      781 taccagaggg cgccccagct ggcaattcct cttaagtaag taagagtata cgtatatcgg
      841 ctaataacgt attaaggcgc ttcggcgcct ttttttatgg gggtattttc atcccaatcc
      901 acacgtccaa cgcacagcaa acaccacgtc gaccctatca gctgcgtgct ttctatgagt
      961 cgttgctgca taacaaaaaa ctgcaaaaaa tagtttgaca ggacacgtgt ccaactttaa
     1021 gatgtaccca aagttggaca cgtgtccaac tttttctaac ccctaccaac tggtcggggt
     1081 ttgaaacaga gacctcgttt acctatcggt ctcatgcttg ggcccgaaca aaaactcatc
     1141 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg
     1201 ctctccagct tgggtgtttt ggcggatgag agaagatttt cagcctgata cagattaaat
     1261 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     1321 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     1381 ctgcccatgc gagagtaggc aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     1441 gactgggcct ttcgttttat ctgttgtttg tcggtgaact ctcggtacca aagacgaaca
     1501 ataagacgct gaaaagcgtc ttttttcgtt ttggtccgct gctctcctga gtaggacaaa
     1561 tccgccgccc tagacctagg gtacgggttt tgctgcccgc aaacgggctg ttctggtgtt
     1621 gctagtttgt tatcagaatc gcagatccgg cttcagccgg tttgccggct gaaagcacta
     1681 tttcttccag aattgccatg attttttccc cacgggaggc gtcactggct cccgtgttgt
     1741 cggcagcttt gattcgataa gcagcatcgc ctgtttcagg ctgtctatgt gtgactgttg
     1801 agctgtaaca agttgtctca ggtgttcaat ttcatgttct agttgctttg ttttactggt
     1861 ttcacctgtt ctattaggtg ttacatgctg ttcatctgtt acattgtcga tctgttcatg
     1921 gtgaacagct ttgaatgcac caaaaactcg taaaagctct gatgtatcta tcttttttac
     1981 accgttttca tctgtgcata tggacagttt tccctttgat atgtaacggt gaacagttgt
     2041 tctacttttg tttgttagtc ttgatgcttc actgatagat acaagagcca taagaacctc
     2101 agatccttcc gtatttagcc agtatgttct ctagtgtggt tcgttgtttt tgcgtgagcc
     2161 atgagaacga accattgaga tcatacttac tttgcatgtc actcaaaaat tttgcctcaa
     2221 aactggtgag ctgaattttt gcagttaaag catcgtgtag tgtttttctt agtccgttat
     2281 gtaggtagga atctgatgta atggttgttg gtattttgtc accattcatt tttatctggt
     2341 tgttctcaag ttcggttacg agatccattt gtctatctag ttcaacttgg aaaatcaacg
     2401 tatcagtcgg gcggcctcgc ttatcaacca ccaatttcat attgctgtaa gtgtttaaat
     2461 ctttacttat tggtttcaaa acccattggt taagcctttt aaactcatgg tagttatttt
     2521 caagcattaa catgaactta aattcatcaa ggctaatctc tatatttgcc ttgtgagttt
     2581 tcttttgtgt tagttctttt aataaccact cataaatcct catagagtat ttgttttcaa
     2641 aagacttaac atgttccaga ttatatttta tgaatttttt taactggaaa agataaggca
     2701 atatctcttc actaaaaact aattctaatt tttcgcttga gaacttggca tagtttgtcc
     2761 actggaaaat ctcaaagcct ttaaccaaag gattcctgat ttccacagtt ctcgtcatca
     2821 gctctctggt tgctttagct aatacaccat aagcattttc cctactgatg ttcatcatct
     2881 gagcgtattg gttataagtg aacgataccg tccgttcttt ccttgtaggg ttttcaatcg
     2941 tggggttgag tagtgccaca cagcataaaa ttagcttggt ttcatgctcc gttaagtcat
     3001 agcgactaat cgctagttca tttgctttga aaacaactaa ttcagacata catctcaatt
     3061 ggtctaggtg attttaatca ctataccaat tgagatgggc tagtcaatga taattactag
     3121 tccttttcct ttgagttgtg ggtatctgta aattctgcta gacctttgct ggaaaacttg
     3181 taaattctgc tataccctct gtaaattccg ctagaccttt gtgtgttttt tttgtttata
     3241 ttcaagtggt tataatttat agaataaaga aagaataaaa aaagataaaa agaatagatc
     3301 ccagccctgt gtataactca ctactttagt cagttccgca gtattacaaa aggatgtcgc
     3361 aaacgctgtt tgctcctcta caaaacagac cttaaaaccc taaaggctta agtagcaccc
     3421 tcgcaagctc gggcaaatcg ctgaatattc cttttgtctc cgaccatcag gcacctgagt
     3481 cgctgtcttt ttcgtgacat tcagttcgct gcgctcacgg ctctggcagt gaatgggggt
     3541 aaatggcact acaggcgcct tttatggatt catgcaagga aactacccat aatacaagaa
     3601 aagcccgtca cgggcttctc agggcgtttt atggcgggtc tgctatgtgg tgctatctga
     3661 ctttttgctg ttcagcagtt cctgccctct gattttccag tctgaccact tcggattatc
     3721 ccgtgacagg tcattcagac tggctaatgc acccagtaag gcagcggtat catcaacagg
     3781 cttacccgtc ttactgtccc tagtggatta tcaaaaagga tcttcaccta gatcctttta
     3841 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3901 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3961 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4021 agtgctgcaa tgataccgcg ggacccacgc tcaccggctc cagatttatc agcaataaac
     4081 cagccagccg gaagggccaa gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4141 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4201 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4261 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4321 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4381 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4441 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4501 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4561 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4621 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4681 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4741 cggaaatgtt gaatactcat actcttcctt
//
