LOCUS       sequence-369235-        6231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc inducible dCasRx-IF3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6231)
  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 6231)
  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: "Nucleic 
            Acids Res. 2022 Aug 11. pii: 6660959. doi: 10.1093/nar/gkac680."
COMMENT     Sequence Label: pPBO.ACT003
FEATURES             Location/Qualifiers
     source          1..6231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(81..92)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(627..3527)
                     /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"
     RBS             3537..3548
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        3566..3621
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    3567..3585
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3637..4263
                     /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
                     VKNKRALXDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(3713..3732)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        4293..4395
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4396..5211
                     /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             4396..5211
                     /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(4469..4488)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      5232..5326
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      5440..5985
                     /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     5886..5905
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6102..6119
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6147..6174)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(join(6190..6231,1..30))
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gtctttcgac tgagcctttc gttttatttg atgccctatt attactgctt ttttttggga
       61 gccaaaacca tgatcatttg gcgaccttca atttttgtag gaaaactctc gactacagct
      121 aactcttgta aatcatcttt tacgcggttc aagacttcca tcccgatttg ttggtgtgcc
      181 atctcgcgac cgcgaaagcg taacgtaatt ttagcmttgt ctccttcctc aagaaaacga
      241 ataagtgaac gcaatttgac ctggtagtcg ccctcatccg tacccgggcg aaatttaatc
      301 tcttttactt gaatcacctt ttgcttcttt ttctgttctt tgcttgattt actcttttca
      361 tacaggaatt taccatagtc catgatgcgg cacacgggtg gctcggcgtt aggactaatc
      421 tctactaagt cgacaccggc ttcttcagct ttttccaacg cctcgcgcaa gctcacgata
      481 ccaagttgct caccctctaa ccccgtcagg cgaacctcct gtgcgcgaat ctcgccgtta
      541 atgcggttag ggcgtgcggt ctgaacgcgt tttcctcctt tcgaccctcc gcctccgcta
      601 ccgcctccac cagagcctcc tccaccggaa ttgccggaca ccttcttttt ctccttgtcg
      661 aacttggcgg cctcgttcct atcgaacagg gcctcgatgc tcaggttctt aaacctgggg
      721 atacagtagc cgaaaggcac acacagcagt ttcaggagcc tatcgttgta cttcttctcg
      781 tcattcacag cgtcgaagta ctcggacacc tttccgctgc ttttctcgta cctctcattc
      841 atgataattc tctgcatgat gtaatggtac agttggaagt aggaattgac ctcggcaatg
      901 tcgttgatat aggcgtggac atacctggcc acttccaggg cgacggcctt gtttgcgaac
      961 agggtacatg tcttgttgtc aattctcagg aggtcctccc tgatgatcac attccacttg
     1021 gtgttcctca ggtaggcgtt cagggcggtt ttggccttct ccctgttaat ctgcctggtg
     1081 aactcctcgg ctttcttctc gtcgctgtat ttgatgtaat tggcatacag cttggggttg
     1141 gcgctctcga ggctgtcaat gctctccttg gctctttcag ccatctcctt ttccacgtcc
     1201 tttctcttat cgggggcagt ttcatcaatg ccagcgcaga gcttggtgac ggagctgaat
     1261 cccttctctt ccagtttctt gagattgatg tcgtagcctt tctccttgta cagttgagca
     1321 tcacgctcga cgcaatggaa tccgatgacg tacctggcgt tgatattgac aatattcttg
     1381 aggatgtggt agatcacggt gaggtacagg ctgatgatct ttttaaactt ctccctctcg
     1441 gcgttttccc tgccggtgtc ctcaatgacg ctccttttct tgtcgaattg gtcgtagttc
     1501 attccggtga tgatctttgt gagagcgtcc accttttcgc tcacgctctt gcccttatcc
     1561 tttccgatac aagtttcgta gtacctgtcg atctggttct tgccgttctg gccctgtttt
     1621 ttctggatgt cagcgatcct gccgagcacg aacttcacca cggcctcgtt tttggcgatc
     1681 tcatggaggt gggcaggatc accgtatctg atcaggtagt ggaacctttt attgctgatc
     1741 acgttattaa taatgaaatt tctcatgccg tgcttgcctt tcttgagctt gtttccgttc
     1801 tcgtccaggg aaaaggtgtc ggcgagggcc ttgagctcat cataggacag gttggttcct
     1861 aaaatacgga tggcgtcgat atacatggcc ctcctggcat cggcaatagg ttctcccatt
     1921 ctagcgaagg acttgatcag cctcagctca tcggcgatct tggcggagtc tttgaaaaag
     1981 gcgtattcct ccacgaactt agcgttgact ccgatgagag gcatcacctt caggaagctc
     2041 tggatgttat cgaatttatt aatcagggtg gtcaggaggt cgttgatctc cttgccatcc
     2101 aggaacatgg tcagggcata catgagtttg ctgaaggcgg aaacatcacg gccagcgggc
     2161 aggattctgg gcagtctagg ggcgtccttc ttcttatact ctcttgtctt gtttccccta
     2221 aattccttga tgttgtgcat gatattttcg agctttctcc aaattctatt agcttcatcg
     2281 tagtagaggg catccttctg gtcgtcgttg aaggagcccc tcaggttaat cacaaagata
     2341 tccttctcgc tcagggactt ctcattatcg gggagggact tattggcggc agccaccttg
     2401 gcatcctctt cgatgtaata cctataaatc acaaagtcca tcatggtgta gaccttggtc
     2461 ctgatggagt cgaacacctt atgatttttc ctgatctcgg acatatcctt cctgtccagc
     2521 atcacttccc tgagcttggt gatattgaat ccgaggtttt tctgctcttt cataatgctg
     2581 aatctgaaat attgttcggc gaattcggca gggttgattc ccagagtttc ggcaatatag
     2641 ttcacgttgg cggcggagtt cttggagaag gagttggtca gctcattggt gatcctgtcg
     2701 tagaggtagt tgagggtgga gatgtattcg ttgtcgaggt tcttatcgag gttgtagagc
     2761 caggtcctgg agatcctgga ctcttcttcg ttgttagcga ccacccagtg cgccagtccg
     2821 ctcaggaggg ccagaatgtc atagcattcg ttgccgtaat tgatgatgta atttctgccc
     2881 tccttgctga aaaaggcctg gccgaaatag ccgagtctgg ggttatcgag gaagttgtcg
     2941 aactcgtcat actgggcctt gatggcgttg atgagcttat cgttattgtt gaaagcggcc
     3001 ctatggtgct cggggtcttt gaattcgtcg taggtataca ctgtggagaa cttgccgaat
     3061 ccaataatgt ccttatccag gccggagata ttgttgacgg cgtaggcggc gttggtaatg
     3121 tattcggcga ggattttttc aatgtccagg atgttatgga tcacctggat acaaatattg
     3181 tcattgccat cagcgctctc gccgaagtac ctcttttcca gagtttcctt caggccgagc
     3241 atatcctgct ggacgggtcc tgtatacaga gggttgttag ccaccacggc gtagccctta
     3301 ggatggctga atttggcgtt gccgatctta tagccggcgt ttttatcggc catttcagcg
     3361 ctgaaggcct cgccctcatt cacgctcctg atgctgtcgc cctccacgat cttttccagc
     3421 ctggcgtcgc tgccttcggc gaaggttgtc atgtacactt tggagccgga cacgagtgtg
     3481 gacttcacgc ccatgccctt ggcgaaggac tttttttttt cgatcatgta agatcctttc
     3541 tcctctttag atcttttgaa ttcttttctc tatcactgat agggagtggt aaaataactc
     3601 tatcaacgat agagtgtcaa caaaaattag gaattaatga tgtctagatt agataaaagt
     3661 aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg tttaacaacc
     3721 cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca tgtaaaaaat
     3781 aagcgggctt tgcycgacgc cttagccatt gagatgttag ataggcacca tactcacttt
     3841 tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa aagttttaga
     3901 tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg gcctacagaa
     3961 aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg tttttcacta
     4021 gagaatgcat tatatgcact cagcgctgtg gggcatttta ctttaggttg cgtattggaa
     4081 gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga tagtatgccg
     4141 ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc agccttctta
     4201 ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga aagtgggtct
     4261 taagacgtcg atatctggcg aaaatgaaac gttgatcggc acgtaagagg ttccaacttt
     4321 caccataatg aaataagatc actaccgggc gtattttttg agttatcgag attttcagga
     4381 gctaaggaag ctaaaatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta
     4441 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     4501 tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa
     4561 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     4621 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     4681 ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat
     4741 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     4801 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     4861 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     4921 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     4981 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     5041 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     5101 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     5161 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atttgatatc
     5221 gagctcgctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt
     5281 gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gcactagtaa
     5341 caacttatat cgtatggggc tgacttcagg tgctacattt gaagagataa attgcactga
     5401 aatctagaaa tattttatct gattaataag atgatcttct tgagatcgtt ttggtctgcg
     5461 cgtaatctct tgctctgaaa acgaaaaaac cgccttgcag ggcggttttt cgaaggttct
     5521 ctgagctacc aactctttga accgaggtaa ctggcttgga ggagcgcagt caccaaaact
     5581 tgtcctttca gtttagcctt aaccggcgca tgacttcaag actaactcct ctaaatcaat
     5641 taccagtggc tgctgccagt ggtgcttttg catgtctttc cgggttggac tcaagacgat
     5701 agttaccgga taaggcgcag cggtcggact gaacgggggg ttcgtgcata cagtccagct
     5761 tggagcgaac tgcctacccg gaactgagtg tcaggcgtgg aatgagacaa acgcggccat
     5821 aacagcggaa tgacaccggt aaaccgaaag gcaggaacag gagagcgcac gagggagccg
     5881 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacca ctgatttgag
     5941 cgtcagattt cgtgatgctt gtcagggggg cggagcctat ggaaaaacgg ctttgccgcg
     6001 gccctctcac ttccctgtta agtatcttcc tggcatcttc caggaaatct ccgccccgtt
     6061 cgtaagccat ttccgctcgc cgcagtcgaa cgaccgagcg tagcgagtca gtgagcgagg
     6121 aagcggaata tatccctagg tataaacgca gaaaggccca cccgaaggtg agccagtgtg
     6181 actctagtag agagcgttca ccgacaaaca acagataaaa cgaaaggccc a
//
