LOCUS       sequence-361292-        5716 bp    DNA     circular SYN 14-APR-2025
DEFINITION  aTc inducible dCasRx.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5716)
  AUTHORS   Otoupal PB, Cress BF, Doudna JA, Schoeniger JS
  TITLE     CRISPR-RNAa: targeted activation of translation using dCas13 fusions
            to translation initiation factors.
  JOURNAL   Nucleic Acids Res. 2022 Aug 11. pii: 6660959. doi: 
            10.1093/nar/gkac680.
  PUBMED    35950485
REFERENCE   2  (bases 1 to 5716)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2022 Aug 11. pii: 6660959. doi: 10.1093/nar/gkac680."
COMMENT     Sequence Label: pPBO.ACT001
FEATURES             Location/Qualifiers
     source          1..5716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             850..861
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        879..934
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    880..898
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             950..1576
                     /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     complement(1026..1045)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        1606..1708
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1709..2524
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             1709..2524
                     /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
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1782..1801)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2545..2639
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      2753..3298
                     /direction=RIGHT
                     /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     3199..3218
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3415..3432
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3460..3487)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(3503..3574)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3602..3649)
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             complement(join(3656..5716,1..840))
                     /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"
ORIGIN
        1 ggtgatcctg tcgtagaggt agttgagggt ggagatgtat tcgttgtcga ggttcttatc
       61 gaggttgtag agccaggtcc tggagatcct ggactcttct tcgttgttag cgaccaccca
      121 gtgcgccagt ccgctcagga gggccagaat gtcatagcat tcgttgccgt aattgatgat
      181 gtaatttctg ccctccttgc tgaaaaaggc ctggccgaaa tagccgagtc tggggttatc
      241 gaggaagttg tcgaactcgt catactgggc cttgatggcg ttgatgagct tatcgttatt
      301 gttgaaagcg gccctatggt gctcggggtc tttgaattcg tcgtaggtat acactgtgga
      361 gaacttgccg aatccaataa tgtccttatc caggccggag atattgttga cggcgtaggc
      421 ggcgttggta atgtattcgg cgaggatttt ttcaatgtcc aggatgttat ggatcacctg
      481 gatacaaata ttgtcattgc catcagcgct ctcgccgaag tacctctttt ccagagtttc
      541 cttcaggccg agcatatcct gctggacggg tcctgtatac agagggttgt tagccaccac
      601 ggcgtagccc ttaggatggc tgaatttggc gttgccgatc ttatagccgg cgtttttatc
      661 ggccatttca gcgctgaagg cctcgccctc attcacgctc ctgatgctgt cgccctccac
      721 gatcttttcc agcctggcgt cgctgccttc ggcgaaggtt gtcatgtaca ctttggagcc
      781 ggacacgagt gtggacttca cgcccatgcc cttggcgaag gacttttttt tttcgatcat
      841 gtaagatcct ttctcctctt tagatctttt gaattctttt ctctatcact gatagggagt
      901 ggtaaaataa ctctatcaac gatagagtgt caacaaaaat taggaattaa tgatgtctag
      961 attagataaa agtaaagtga ttaacagcgc attagagctg cttaatgagg tcggaatcga
     1021 aggtttaaca acccgtaaac tcgcccagaa gctaggtgta gagcagccta cattgtattg
     1081 gcatgtaaaa aataagcggg ctttgctcga cgccttagcc attgagatgt tagataggca
     1141 ccatactcac ttttgccctt tagaagggga aagctggcaa gattttttac gtaataacgc
     1201 taaaagtttt agatgtgctt tactaagtca tcgcgatgga gcaaaagtac atttaggtac
     1261 acggcctaca gaaaaacagt atgaaactct cgaaaatcaa ttagcctttt tatgccaaca
     1321 aggtttttca ctagagaatg cattatatgc actcagcgct gtggggcatt ttactttagg
     1381 ttgcgtattg gaagatcaag agcatcaagt cgctaaagaa gaaagggaaa cacctactac
     1441 tgatagtatg ccgccattat tacgacaagc tatcgaatta tttgatcacc aaggtgcaga
     1501 gccagccttc ttattcggcc ttgaattgat catatgcgga ttagaaaaac aacttaaatg
     1561 tgaaagtggg tcttaagacg tcgatatctg gcgaaaatga aacgttgatc ggcacgtaag
     1621 aggttccaac tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttatc
     1681 gagattttca ggagctaagg aagctaaaat gagccatatt caacgggaaa cgtcttgctc
     1741 taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     1801 taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga
     1861 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     1921 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     1981 tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga
     2041 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     2101 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     2161 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     2221 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     2281 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     2341 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     2401 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     2461 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     2521 ctaatttgat atcgagctcg cttggactcc tgttgataga tccagtaatg acctcagaac
     2581 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc
     2641 caagcactag taacaactta tatcgtatgg ggctgacttc aggtgctaca tttgaagaga
     2701 taaattgcac tgaaatctag aaatatttta tctgattaat aagatgatct tcttgagatc
     2761 gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg cagggcggtt
     2821 tttcgaaggt tctctgagct accaactctt tgaaccgagg taactggctt ggaggagcgc
     2881 agtcaccaaa acttgtcctt tcagtttagc cttaaccggc gcatgacttc aagactaact
     2941 cctctaaatc aattaccagt ggctgctgcc agtggtgctt ttgcatgtct ttccgggttg
     3001 gactcaagac gatagttacc ggataaggcg cagcggtcgg actgaacggg gggttcgtgc
     3061 atacagtcca gcttggagcg aactgcctac ccggaactga gtgtcaggcg tggaatgaga
     3121 caaacgcggc cataacagcg gaatgacacc ggtaaaccga aaggcaggaa caggagagcg
     3181 cacgagggag ccgccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3241 ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc tatggaaaaa
     3301 cggctttgcc gcggccctct cacttccctg ttaagtatct tcctggcatc ttccaggaaa
     3361 tctccgcccc gttcgtaagc catttccgct cgccgcagtc gaacgaccga gcgtagcgag
     3421 tcagtgagcg aggaagcgga atatatccct aggtataaac gcagaaaggc ccacccgaag
     3481 gtgagccagt gtgactctag tagagagcgt tcaccgacaa acaacagata aaacgaaagg
     3541 cccagtcttt cgactgagcc tttcgtttta tttgatgccc tatgagacct tattagctag
     3601 ctttatcatc atcatcttta taatctttat catcatcatc tttataatcc tcgagggaat
     3661 tgccggacac cttctttttc tccttgtcga acttggcggc ctcgttccta tcgaacaggg
     3721 cctcgatgct caggttctta aacctgggga tacagtagcc gaaaggcaca cacagcagtt
     3781 tcaggagcct atcgttgtac ttcttctcgt cattcacagc gtcgaagtac tcggacacct
     3841 ttccgctgct tttctcgtac ctctcattca tgataattct ctgcatgatg taatggtaca
     3901 gttggaagta ggaattgacc tcggcaatgt cgttgatata ggcgtggaca tacctggcca
     3961 cttccagggc gacggccttg tttgcgaaca gggtacatgt cttgttgtca attctcagga
     4021 ggtcctccct gatgatcaca ttccacttgg tgttcctcag gtaggcgttc agggcggttt
     4081 tggccttctc cctgttaatc tgcctggtga actcctcggc tttcttctcg tcgctgtatt
     4141 tgatgtaatt ggcatacagc ttggggttgg cgctctcgag gctgtcaatg ctctccttgg
     4201 ctctttcagc catctccttt tccacgtcct ttctcttatc gggggcagtt tcatcaatgc
     4261 cagcgcagag cttggtgacg gagctgaatc ccttctcttc cagtttcttg agattgatgt
     4321 cgtagccttt ctccttgtac agttgagcat cacgctcgac gcaatggaat ccgatgacgt
     4381 acctggcgtt gatattgaca atattcttga ggatgtggta gatcacggtg aggtacaggc
     4441 tgatgatctt tttaaacttc tccctctcgg cgttttccct gccggtgtcc tcaatgacgc
     4501 tccttttctt gtcgaattgg tcgtagttca ttccggtgat gatctttgtg agagcgtcca
     4561 ccttttcgct cacgctcttg cccttatcct ttccgataca agtttcgtag tacctgtcga
     4621 tctggttctt gccgttctgg ccctgttttt tctggatgtc agcgatcctg ccgagcacga
     4681 acttcaccac ggcctcgttt ttggcgatct catggaggtg ggcaggatca ccgtatctga
     4741 tcaggtagtg gaacctttta ttgctgatca cgttattaat aatgaaattt ctcatgccgt
     4801 gcttgccttt cttgagcttg tttccgttct cgtccaggga aaaggtgtcg gcgagggcct
     4861 tgagctcatc ataggacagg ttggttccta aaatacggat ggcgtcgata tacatggccc
     4921 tcctggcatc ggcaataggt tctcccattc tagcgaagga cttgatcagc ctcagctcat
     4981 cggcgatctt ggcggagtct ttgaaaaagg cgtattcctc cacgaactta gcgttgactc
     5041 cgatgagagg catcaccttc aggaagctct ggatgttatc gaatttatta atcagggtgg
     5101 tcaggaggtc gttgatctcc ttgccatcca ggaacatggt cagggcatac atgagtttgc
     5161 tgaaggcgga aacatcacgg ccagcgggca ggattctggg cagtctaggg gcgtccttct
     5221 tcttatactc tcttgtcttg tttcccctaa attccttgat gttgtgcatg atattttcga
     5281 gctttctcca aattctatta gcttcatcgt agtagagggc atccttctgg tcgtcgttga
     5341 aggagcccct caggttaatc acaaagatat ccttctcgct cagggacttc tcattatcgg
     5401 ggagggactt attggcggca gccaccttgg catcctcttc gatgtaatac ctataaatca
     5461 caaagtccat catggtgtag accttggtcc tgatggagtc gaacacctta tgatttttcc
     5521 tgatctcgga catatccttc ctgtccagca tcacttccct gagcttggtg atattgaatc
     5581 cgaggttttt ctgctctttc ataatgctga atctgaaata ttgttcggcg aattcggcag
     5641 ggttgattcc cagagtttcg gcaatatagt tcacgttggc ggcggagttc ttggagaagg
     5701 agttggtcag ctcatt
//
