LOCUS       sequence-467616-        6367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi-ART plasmid encoding aTc-inducible dRfxCas13d and 
            constitutive crRNA with negative control (RFP-targeting) spacer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6367)
  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 6367)
  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: pBFC0993
FEATURES             Location/Qualifiers
     source          1..6367
                     /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             298..327
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             343..360
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(416..433)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      586..632
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(670..1296)
                     /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     1201..1220
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        1312..1367
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1348..1366
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1385..1396
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1406..4306
                     /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(4482..4516)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(4587..5246)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5161..5180
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(5485..5502)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5619..6164)
                     /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(5699..5718)
                     /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 aaacaactct ttgataacgt cttcgctact cgccaatgct tgggcccgaa
      301 caaaaactca tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat
      361 tgagtttaaa cgctctccag cttgggtgtt ttggcggatg agagaagatt ttcagcctga
      421 tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta
      481 gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg
      541 gtagtgtggg gtctgcccat gcgagagtag gcaactgcca ggcatcaaat aaaacgaaag
      601 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa ctcgccagat
      661 atcgacgtct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa
      721 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg
      781 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg
      841 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata
      901 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt
      961 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact
     1021 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc
     1081 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag
     1141 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg
     1201 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct
     1261 gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt tgttgacact
     1321 ctatcgttga tagagttatt ttaccactcc ctatcagtga tagagaaaag aattcaaaag
     1381 atctaaagag gagaaaggat cttacatgat cgaaaaaaaa aagtccttcg ccaagggcat
     1441 gggcgtgaag tccacactcg tgtccggctc caaagtgtac atgacaacct tcgccgaagg
     1501 cagcgacgcc aggctggaaa agatcgtgga gggcgacagc atcaggagcg tgaatgaggg
     1561 cgaggccttc agcgctgaaa tggccgataa aaacgccggc tataagatcg gcaacgccaa
     1621 attcagccat cctaagggct acgccgtggt ggctaacaac cctctgtata caggacccgt
     1681 ccagcaggat atgctcggcc tgaaggaaac tctggaaaag aggtacttcg gcgagagcgc
     1741 tgatggcaat gacaatattt gtatccaggt gatccataac atcctggaca ttgaaaaaat
     1801 cctcgccgaa tacattacca acgccgccta cgccgtcaac aatatctccg gcctggataa
     1861 ggacattatt ggattcggca agttctccac agtgtatacc tacgacgaat tcaaagaccc
     1921 cgagcaccat agggccgctt tcaacaataa cgataagctc atcaacgcca tcaaggccca
     1981 gtatgacgag ttcgacaact tcctcgataa ccccagactc ggctatttcg gccaggcctt
     2041 tttcagcaag gagggcagaa attacatcat caattacggc aacgaatgct atgacattct
     2101 ggccctcctg agcggactgg cgcactgggt ggtcgctaac aacgaagaag agtccaggat
     2161 ctccaggacc tggctctaca acctcgataa gaacctcgac aacgaataca tctccaccct
     2221 caactacctc tacgacagga tcaccaatga gctgaccaac tccttctcca agaactccgc
     2281 cgccaacgtg aactatattg ccgaaactct gggaatcaac cctgccgaat tcgccgaaca
     2341 atatttcaga ttcagcatta tgaaagagca gaaaaacctc ggattcaata tcaccaagct
     2401 cagggaagtg atgctggaca ggaaggatat gtccgagatc aggaaaaatc ataaggtgtt
     2461 cgactccatc aggaccaagg tctacaccat gatggacttt gtgatttata ggtattacat
     2521 cgaagaggat gccaaggtgg ctgccgccaa taagtccctc cccgataatg agaagtccct
     2581 gagcgagaag gatatctttg tgattaacct gaggggctcc ttcaacgacg accagaagga
     2641 tgccctctac tacgatgaag ctaatagaat ttggagaaag ctcgaaaata tcatgcacaa
     2701 catcaaggaa tttaggggaa acaagacaag agagtataag aagaaggacg cccctagact
     2761 gcccagaatc ctgcccgctg gccgtgatgt ttccgccttc agcaaactca tgtatgccct
     2821 gaccatgttc ctggatggca aggagatcaa cgacctcctg accaccctga ttaataaatt
     2881 cgataacatc cagagcttcc tgaaggtgat gcctctcatc ggagtcaacg ctaagttcgt
     2941 ggaggaatac gcctttttca aagactccgc caagatcgcc gatgagctga ggctgatcaa
     3001 gtccttcgct agaatgggag aacctattgc cgatgccagg agggccatgt atatcgacgc
     3061 catccgtatt ttaggaacca acctgtccta tgatgagctc aaggccctcg ccgacacctt
     3121 ttccctggac gagaacggaa acaagctcaa gaaaggcaag cacggcatga gaaatttcat
     3181 tattaataac gtgatcagca ataaaaggtt ccactacctg atcagatacg gtgatcctgc
     3241 ccacctccat gagatcgcca aaaacgaggc cgtggtgaag ttcgtgctcg gcaggatcgc
     3301 tgacatccag aaaaaacagg gccagaacgg caagaaccag atcgacaggt actacgaaac
     3361 ttgtatcgga aaggataagg gcaagagcgt gagcgaaaag gtggacgctc tcacaaagat
     3421 catcaccgga atgaactacg accaattcga caagaaaagg agcgtcattg aggacaccgg
     3481 cagggaaaac gccgagaggg agaagtttaa aaagatcatc agcctgtacc tcaccgtgat
     3541 ctaccacatc ctcaagaata ttgtcaatat caacgccagg tacgtcatcg gattccattg
     3601 cgtcgagcgt gatgctcaac tgtacaagga gaaaggctac gacatcaatc tcaagaaact
     3661 ggaagagaag ggattcagct ccgtcaccaa gctctgcgct ggcattgatg aaactgcccc
     3721 cgataagaga aaggacgtgg aaaaggagat ggctgaaaga gccaaggaga gcattgacag
     3781 cctcgagagc gccaacccca agctgtatgc caattacatc aaatacagcg acgagaagaa
     3841 agccgaggag ttcaccaggc agattaacag ggagaaggcc aaaaccgccc tgaacgccta
     3901 cctgaggaac accaagtgga atgtgatcat cagggaggac ctcctgagaa ttgacaacaa
     3961 gacatgtacc ctgttcgcaa acaaggccgt cgccctggaa gtggccaggt atgtccacgc
     4021 ctatatcaac gacattgccg aggtcaattc ctacttccaa ctgtaccatt acatcatgca
     4081 gagaattatc atgaatgaga ggtacgagaa aagcagcgga aaggtgtccg agtacttcga
     4141 cgctgtgaat gacgagaaga agtacaacga taggctcctg aaactgctgt gtgtgccttt
     4201 cggctactgt atccccaggt ttaagaacct gagcatcgag gccctgttcg ataggaacga
     4261 ggccgccaag ttcgacaagg agaaaaagaa ggtgtccggc aattcctaat aaatccctcg
     4321 gtaccaaaga cgaacaataa gacgctgaaa agcgtctttt ttcgttttgg tccgctgagc
     4381 agttacagag atgttacgaa ccactagtgc actgcagtac acctttggtc gaaaaaaaag
     4441 cccgcactgt caggtgcggg cttttttctg tgtttccacc aataaaaaac gcccggcggc
     4501 aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca
     4561 ggagtccaag cgagctcgat atcaaattac gccccgccct gccactcatc gcagtactgt
     4621 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
     4681 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg
     4741 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
     4801 ggattggctg aaacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
     4861 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     4921 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     4981 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacgaaa ttccggatga
     5041 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc
     5101 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     5161 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     5221 gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac
     5281 tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg
     5341 tgcccgatca agcgctagcg gagtgtatac tggcttacta tgttggcact gatgagggtg
     5401 tcagtgaagt gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat
     5461 gtgatacagg atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact
     5521 gcggcgagcg gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat
     5581 acttaacagg gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct
     5641 gacaagcatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa
     5701 agataccagg cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg
     5761 tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag
     5821 ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga
     5881 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc
     5941 accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg
     6001 gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc
     6061 ggttcaaaga gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt
     6121 cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta
     6181 atcagataaa atatttctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     6241 tcagccccat acgatataag ttgtttcagc caaaaaactt aagaccgccg gtcttgtcca
     6301 ctaccttgca gtaatgcggt ggacaggatc ggcggttttc ttttctcttc tcaaccctga
     6361 attcgca
//
