LOCUS       sequence-467618-        6362 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi-ART plasmid encoding aTc-inducible dRfxCas13d and 
            constitutive crRNA with 2xBsaI spacer cloning site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6362)
  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 6362)
  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: pBFC0984
FEATURES             Location/Qualifiers
     source          1..6362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(138..181)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        190..224
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        225..254
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     CDS             293..322
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             338..355
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(411..428)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      581..627
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(665..1291)
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     1196..1215
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        1307..1362
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1343..1361
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1380..1391
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1401..4301
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS
                     IRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLE
                     KRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTV
                     YTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYII
                     NYGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITN
                     ELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRK
                     DMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFV
                     INLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPA
                     GRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAF
                     FKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDE
                     NGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQ
                     KKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRE
                     NAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLE
                     EKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKK
                     AEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVH
                     AYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCV
                     PFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     terminator      complement(4477..4511)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(4582..5241)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5156..5175
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(5480..5497)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5614..6159)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(5694..5713)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctagactga actggccgat aattgcagac ggtttaaaca tccctcggta ccaaattcca
       61 gaaaagaggc ctcccgaaag gggggccttt tttcgttttg gtccgctgcc aaaccagatg
      121 tcaacacagc tacaacgatt tcagataaaa aaaatcctta gctttcgcta aggatgattt
      181 ctggaattct tgacagctag ctcagtccta ggtataatac tagtaacccc taccaactgg
      241 tcggggtttg aaacagagac ctcgtttacc tatcggtctc atgcttgggc ccgaacaaaa
      301 actcatctca gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcattgagt
      361 ttaaacgctc tccagcttgg gtgttttggc ggatgagaga agattttcag cctgatacag
      421 attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg
      481 gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt
      541 gtggggtctg cccatgcgag agtaggcaac tgccaggcat caaataaaac gaaaggctca
      601 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaactcgc cagatatcga
      661 cgtcttaaga cccactttca catttaagtt gtttttctaa tccgcatatg atcaattcaa
      721 ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa taattcgata gcttgtcgta
      781 ataatggcgg catactatca gtagtaggtg tttccctttc ttctttagcg acttgatgct
      841 cttgatcttc caatacgcaa cctaaagtaa aatgccccac agcgctgagt gcatataatg
      901 cattctctag tgaaaaacct tgttggcata aaaaggctaa ttgattttcg agagtttcat
      961 actgtttttc tgtaggccgt gtacctaaat gtacttttgc tccatcgcga tgacttagta
     1021 aagcacatct aaaactttta gcgttattac gtaaaaaatc ttgccagctt tccccttcta
     1081 aagggcaaaa gtgagtatgg tgcctatcta acatctcaat ggctaaggcg tcgagcaaag
     1141 cccgcttatt ttttacatgc caatacaatg taggctgctc tacacctagc ttctgggcga
     1201 gtttacgggt tgttaaacct tcgattccga cctcattaag cagctctaat gcgctgttaa
     1261 tcactttact tttatctaat ctagacatca ttaattccta atttttgttg acactctatc
     1321 gttgatagag ttattttacc actccctatc agtgatagag aaaagaattc aaaagatcta
     1381 aagaggagaa aggatcttac atgatcgaaa aaaaaaagtc cttcgccaag ggcatgggcg
     1441 tgaagtccac actcgtgtcc ggctccaaag tgtacatgac aaccttcgcc gaaggcagcg
     1501 acgccaggct ggaaaagatc gtggagggcg acagcatcag gagcgtgaat gagggcgagg
     1561 ccttcagcgc tgaaatggcc gataaaaacg ccggctataa gatcggcaac gccaaattca
     1621 gccatcctaa gggctacgcc gtggtggcta acaaccctct gtatacagga cccgtccagc
     1681 aggatatgct cggcctgaag gaaactctgg aaaagaggta cttcggcgag agcgctgatg
     1741 gcaatgacaa tatttgtatc caggtgatcc ataacatcct ggacattgaa aaaatcctcg
     1801 ccgaatacat taccaacgcc gcctacgccg tcaacaatat ctccggcctg gataaggaca
     1861 ttattggatt cggcaagttc tccacagtgt atacctacga cgaattcaaa gaccccgagc
     1921 accatagggc cgctttcaac aataacgata agctcatcaa cgccatcaag gcccagtatg
     1981 acgagttcga caacttcctc gataacccca gactcggcta tttcggccag gcctttttca
     2041 gcaaggaggg cagaaattac atcatcaatt acggcaacga atgctatgac attctggccc
     2101 tcctgagcgg actggcgcac tgggtggtcg ctaacaacga agaagagtcc aggatctcca
     2161 ggacctggct ctacaacctc gataagaacc tcgacaacga atacatctcc accctcaact
     2221 acctctacga caggatcacc aatgagctga ccaactcctt ctccaagaac tccgccgcca
     2281 acgtgaacta tattgccgaa actctgggaa tcaaccctgc cgaattcgcc gaacaatatt
     2341 tcagattcag cattatgaaa gagcagaaaa acctcggatt caatatcacc aagctcaggg
     2401 aagtgatgct ggacaggaag gatatgtccg agatcaggaa aaatcataag gtgttcgact
     2461 ccatcaggac caaggtctac accatgatgg actttgtgat ttataggtat tacatcgaag
     2521 aggatgccaa ggtggctgcc gccaataagt ccctccccga taatgagaag tccctgagcg
     2581 agaaggatat ctttgtgatt aacctgaggg gctccttcaa cgacgaccag aaggatgccc
     2641 tctactacga tgaagctaat agaatttgga gaaagctcga aaatatcatg cacaacatca
     2701 aggaatttag gggaaacaag acaagagagt ataagaagaa ggacgcccct agactgccca
     2761 gaatcctgcc cgctggccgt gatgtttccg ccttcagcaa actcatgtat gccctgacca
     2821 tgttcctgga tggcaaggag atcaacgacc tcctgaccac cctgattaat aaattcgata
     2881 acatccagag cttcctgaag gtgatgcctc tcatcggagt caacgctaag ttcgtggagg
     2941 aatacgcctt tttcaaagac tccgccaaga tcgccgatga gctgaggctg atcaagtcct
     3001 tcgctagaat gggagaacct attgccgatg ccaggagggc catgtatatc gacgccatcc
     3061 gtattttagg aaccaacctg tcctatgatg agctcaaggc cctcgccgac accttttccc
     3121 tggacgagaa cggaaacaag ctcaagaaag gcaagcacgg catgagaaat ttcattatta
     3181 ataacgtgat cagcaataaa aggttccact acctgatcag atacggtgat cctgcccacc
     3241 tccatgagat cgccaaaaac gaggccgtgg tgaagttcgt gctcggcagg atcgctgaca
     3301 tccagaaaaa acagggccag aacggcaaga accagatcga caggtactac gaaacttgta
     3361 tcggaaagga taagggcaag agcgtgagcg aaaaggtgga cgctctcaca aagatcatca
     3421 ccggaatgaa ctacgaccaa ttcgacaaga aaaggagcgt cattgaggac accggcaggg
     3481 aaaacgccga gagggagaag tttaaaaaga tcatcagcct gtacctcacc gtgatctacc
     3541 acatcctcaa gaatattgtc aatatcaacg ccaggtacgt catcggattc cattgcgtcg
     3601 agcgtgatgc tcaactgtac aaggagaaag gctacgacat caatctcaag aaactggaag
     3661 agaagggatt cagctccgtc accaagctct gcgctggcat tgatgaaact gcccccgata
     3721 agagaaagga cgtggaaaag gagatggctg aaagagccaa ggagagcatt gacagcctcg
     3781 agagcgccaa ccccaagctg tatgccaatt acatcaaata cagcgacgag aagaaagccg
     3841 aggagttcac caggcagatt aacagggaga aggccaaaac cgccctgaac gcctacctga
     3901 ggaacaccaa gtggaatgtg atcatcaggg aggacctcct gagaattgac aacaagacat
     3961 gtaccctgtt cgcaaacaag gccgtcgccc tggaagtggc caggtatgtc cacgcctata
     4021 tcaacgacat tgccgaggtc aattcctact tccaactgta ccattacatc atgcagagaa
     4081 ttatcatgaa tgagaggtac gagaaaagca gcggaaaggt gtccgagtac ttcgacgctg
     4141 tgaatgacga gaagaagtac aacgataggc tcctgaaact gctgtgtgtg cctttcggct
     4201 actgtatccc caggtttaag aacctgagca tcgaggccct gttcgatagg aacgaggccg
     4261 ccaagttcga caaggagaaa aagaaggtgt ccggcaattc ctaataaatc cctcggtacc
     4321 aaagacgaac aataagacgc tgaaaagcgt cttttttcgt tttggtccgc tgagcagtta
     4381 cagagatgtt acgaaccact agtgcactgc agtacacctt tggtcgaaaa aaaagcccgc
     4441 actgtcaggt gcgggctttt ttctgtgttt ccaccaataa aaaacgcccg gcggcaaccg
     4501 agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat caacaggagt
     4561 ccaagcgagc tcgatatcaa attacgcccc gccctgccac tcatcgcagt actgttgtaa
     4621 ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc
     4681 cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc
     4741 gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt
     4801 ggctgaaacg aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc
     4861 gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc
     4921 actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac
     4981 actatcccat atcaccagct caccgtcttt cattgccata cgaaattccg gatgagcatt
     5041 catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac
     5101 ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac
     5161 tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata
     5221 tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa
     5281 aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccc
     5341 gatcaagcgc tagcggagtg tatactggct tactatgttg gcactgatga gggtgtcagt
     5401 gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat
     5461 acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc
     5521 gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta
     5581 acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa
     5641 gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata
     5701 ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac
     5761 cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg
     5821 ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct
     5881 gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac
     5941 tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa
     6001 ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc
     6061 aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt
     6121 tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag
     6181 ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
     6241 cccatacgat ataagttgtt tcagccaaaa aacttaagac cgccggtctt gtccactacc
     6301 ttgcagtaat gcggtggaca ggatcggcgg ttttcttttc tcttctcaac cctgaattcg
     6361 ca
//
