LOCUS       sequence-366427-        5907 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc inducible dCasRx-IF1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5907)
  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 5907)
  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.ACT002
FEATURES             Location/Qualifiers
     source          1..5907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(59..78)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      822..916
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1030..1575
                     /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     1476..1495
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1692..1709
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(1737..1764)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(1780..1851)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2124..5024)
                     /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             5034..5045
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        5063..5118
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    5064..5082
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             5134..5760
                     /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(5210..5229)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        5790..5892
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(5893..5907,1..801)
                     /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             join(5893..5907,1..801)
                     /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"
ORIGIN
        1 cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg
       61 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg
      121 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
      181 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
      241 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca
      301 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
      361 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
      421 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
      481 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
      541 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
      601 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
      661 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
      721 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
      781 atgctcgatg agtttttcta atttgatatc gagctcgctt ggactcctgt tgatagatcc
      841 agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt
      901 ttttattggt gagaatccaa gcactagtaa caacttatat cgtatggggc tgacttcagg
      961 tgctacattt gaagagataa attgcactga aatctagaaa tattttatct gattaataag
     1021 atgatcttct tgagatcgtt ttggtctgcg cgtaatctct tgctctgaaa acgaaaaaac
     1081 cgccttgcag ggcggttttt cgaaggttct ctgagctacc aactctttga accgaggtaa
     1141 ctggcttgga ggagcgcagt caccaaaact tgtcctttca gtttagcctt aaccggcgca
     1201 tgacttcaag actaactcct ctaaatcaat taccagtggc tgctgccagt ggtgcttttg
     1261 catgtctttc cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcggact
     1321 gaacgggggg ttcgtgcata cagtccagct tggagcgaac tgcctacccg gaactgagtg
     1381 tcaggcgtgg aatgagacaa acgcggccat aacagcggaa tgacaccggt aaaccgaaag
     1441 gcaggaacag gagagcgcac gagggagccg ccagggggaa acgcctggta tctttatagt
     1501 cctgtcgggt ttcgccacca ctgatttgag cgtcagattt cgtgatgctt gtcagggggg
     1561 cggagcctat ggaaaaacgg ctttgccgcg gccctctcac ttccctgtta agtatcttcc
     1621 tggcatcttc caggaaatct ccgccccgtt cgtaagccat ttccgctcgc cgcagtcgaa
     1681 cgaccgagcg tagcgagtca gtgagcgagg aagcggaata tatccctagg tataaacgca
     1741 gaaaggccca cccgaaggtg agccagtgtg actctagtag agagcgttca ccgacaaaca
     1801 acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgccctat
     1861 tattacttga agcgatatgt gatgcgccca cgcgtcaaat cataggggct catttcaacc
     1921 gtcaccttgt caccgacaag gatacggata tagtttttgc ggattttccc gcttacggtc
     1981 gctaaaatct gatgcccgtt ctcaagttcc actgtaaaca tagcgttggg catggtttct
     2041 acaactttgc cttcaatttc gatgacatct tctttggccg accctccgcc tccgctaccg
     2101 cctccaccag agcctcctcc accggaattg ccggacacct tctttttctc cttgtcgaac
     2161 ttggcggcct cgttcctatc gaacagggcc tcgatgctca ggttcttaaa cctggggata
     2221 cagtagccga aaggcacaca cagcagtttc aggagcctat cgttgtactt cttctcgtca
     2281 ttcacagcgt cgaagtactc ggacaccttt ccgctgcttt tctcgtacct ctcattcatg
     2341 ataattctct gcatgatgta atggtacagt tggaagtagg aattgacctc ggcaatgtcg
     2401 ttgatatagg cgtggacata cctggccact tccagggcga cggccttgtt tgcgaacagg
     2461 gtacatgtct tgttgtcaat tctcaggagg tcctccctga tgatcacatt ccacttggtg
     2521 ttcctcaggt aggcgttcag ggcggttttg gccttctccc tgttaatctg cctggtgaac
     2581 tcctcggctt tcttctcgtc gctgtatttg atgtaattgg catacagctt ggggttggcg
     2641 ctctcgaggc tgtcaatgct ctccttggct ctttcagcca tctccttttc cacgtccttt
     2701 ctcttatcgg gggcagtttc atcaatgcca gcgcagagct tggtgacgga gctgaatccc
     2761 ttctcttcca gtttcttgag attgatgtcg tagcctttct ccttgtacag ttgagcatca
     2821 cgctcgacgc aatggaatcc gatgacgtac ctggcgttga tattgacaat attcttgagg
     2881 atgtggtaga tcacggtgag gtacaggctg atgatctttt taaacttctc cctctcggcg
     2941 ttttccctgc cggtgtcctc aatgacgctc cttttcttgt cgaattggtc gtagttcatt
     3001 ccggtgatga tctttgtgag agcgtccacc ttttcgctca cgctcttgcc cttatccttt
     3061 ccgatacaag tttcgtagta cctgtcgatc tggttcttgc cgttctggcc ctgttttttc
     3121 tggatgtcag cgatcctgcc gagcacgaac ttcaccacgg cctcgttttt ggcgatctca
     3181 tggaggtggg caggatcacc gtatctgatc aggtagtgga accttttatt gctgatcacg
     3241 ttattaataa tgaaatttct catgccgtgc ttgcctttct tgagcttgtt tccgttctcg
     3301 tccagggaaa aggtgtcggc gagggccttg agctcatcat aggacaggtt ggttcctaaa
     3361 atacggatgg cgtcgatata catggccctc ctggcatcgg caataggttc tcccattcta
     3421 gcgaaggact tgatcagcct cagctcatcg gcgatcttgg cggagtcttt gaaaaaggcg
     3481 tattcctcca cgaacttagc gttgactccg atgagaggca tcaccttcag gaagctctgg
     3541 atgttatcga atttattaat cagggtggtc aggaggtcgt tgatctcctt gccatccagg
     3601 aacatggtca gggcatacat gagtttgctg aaggcggaaa catcacggcc agcgggcagg
     3661 attctgggca gtctaggggc gtccttcttc ttatactctc ttgtcttgtt tcccctaaat
     3721 tccttgatgt tgtgcatgat attttcgagc tttctccaaa ttctattagc ttcatcgtag
     3781 tagagggcat ccttctggtc gtcgttgaag gagcccctca ggttaatcac aaagatatcc
     3841 ttctcgctca gggacttctc attatcgggg agggacttat tggcggcagc caccttggca
     3901 tcctcttcga tgtaatacct ataaatcaca aagtccatca tggtgtagac cttggtcctg
     3961 atggagtcga acaccttatg atttttcctg atctcggaca tatccttcct gtccagcatc
     4021 acttccctga gcttggtgat attgaatccg aggtttttct gctctttcat aatgctgaat
     4081 ctgaaatatt gttcggcgaa ttcggcaggg ttgattccca gagtttcggc aatatagttc
     4141 acgttggcgg cggagttctt ggagaaggag ttggtcagct cattggtgat cctgtcgtag
     4201 aggtagttga gggtggagat gtattcgttg tcgaggttct tatcgaggtt gtagagccag
     4261 gtcctggaga tcctggactc ttcttcgttg ttagcgacca cccagtgcgc cagtccgctc
     4321 aggagggcca gaatgtcata gcattcgttg ccgtaattga tgatgtaatt tctgccctcc
     4381 ttgctgaaaa aggcctggcc gaaatagccg agtctggggt tatcgaggaa gttgtcgaac
     4441 tcgtcatact gggccttgat ggcgttgatg agcttatcgt tattgttgaa agcggcccta
     4501 tggtgctcgg ggtctttgaa ttcgtcgtag gtatacactg tggagaactt gccgaatcca
     4561 ataatgtcct tatccaggcc ggagatattg ttgacggcgt aggcggcgtt ggtaatgtat
     4621 tcggcgagga ttttttcaat gtccaggatg ttatggatca cctggataca aatattgtca
     4681 ttgccatcag cgctctcgcc gaagtacctc ttttccagag tttccttcag gccgagcata
     4741 tcctgctgga cgggtcctgt atacagaggg ttgttagcca ccacggcgta gcccttagga
     4801 tggctgaatt tggcgttgcc gatcttatag ccggcgtttt tatcggccat ttcagcgctg
     4861 aaggcctcgc cctcattcac gctcctgatg ctgtcgccct ccacgatctt ttccagcctg
     4921 gcgtcgctgc cttcggcgaa ggttgtcatg tacactttgg agccggacac gagtgtggac
     4981 ttcacgccca tgcccttggc gaaggacttt tttttttcga tcatgtaaga tcctttctcc
     5041 tctttagatc ttttgaattc ttttctctat cactgatagg gagtggtaaa ataactctat
     5101 caacgataga gtgtcaacaa aaattaggaa ttaatgatgt ctagattaga taaaagtaaa
     5161 gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt
     5221 aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt aaaaaataag
     5281 cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac tcacttttgc
     5341 cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag ttttagatgt
     5401 gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc tacagaaaaa
     5461 cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt ttcactagag
     5521 aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt attggaagat
     5581 caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag tatgccgcca
     5641 ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc cttcttattc
     5701 ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag tgggtcttaa
     5761 gacgtcgata tctggcgaaa atgaaacgtt gatcggcacg taagaggttc caactttcac
     5821 cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt ttcaggagct
     5881 aaggaagcta aaatgagcca tattcaa
//
