LOCUS       sequence-448801-        5496 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dRxCas13d expressed from pTet promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5496)
  AUTHORS   Cardiff RAL, Faulkner ID, Beall JG, Carothers JM, Zalatan JG
  TITLE     
  JOURNAL   Nucleic Acids Res. 2024 May 22;52(9):5406-5419. doi: 
            10.1093/nar/gkae275.
  PUBMED    38613390
REFERENCE   2  (bases 1 to 5496)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2024-05-22- 22"; volume: "52"; issue: 
            "9"; pages: "5406-5419"
COMMENT     Sequence Label: pRC055
FEATURES             Location/Qualifiers
     source          1..5496
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(32..658)
                     /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     563..582
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        674..729
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    710..728
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             741..752
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             759..3659
                     /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      3686..3757
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3773..3800
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(3828..3845)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3962..4507)
                     /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(4042..4061)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4621..4715)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4736..5395)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5310..5329
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(5396..5496,1..2))
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 caacgtctca ttttcgccag atatcgacgt cttaagaccc actttcacat ttaagttgtt
       61 tttctaatcc gcatatgatc aattcaaggc cgaataagaa ggctggctct gcaccttggt
      121 gatcaaataa ttcgatagct tgtcgtaata atggcggcat actatcagta gtaggtgttt
      181 ccctttcttc tttagcgact tgatgctctt gatcttccaa tacgcaacct aaagtaaaat
      241 gccccacagc gctgagtgca tataatgcat tctctagtga aaaaccttgt tggcataaaa
      301 aggctaattg attttcgaga gtttcatact gtttttctgt aggccgtgta cctaaatgta
      361 cttttgctcc atcgcgatga cttagtaaag cacatctaaa acttttagcg ttattacgta
      421 aaaaatcttg ccagctttcc ccttctaaag ggcaaaagtg agtatggtgc ctatctaaca
      481 tctcaatggc taaggcgtcg agcaaagccc gcttattttt tacatgccaa tacaatgtag
      541 gctgctctac acctagcttc tgggcgagtt tacgggttgt taaaccttcg attccgacct
      601 cattaagcag ctctaatgcg ctgttaatca ctttactttt atctaatcta gacatcatta
      661 attcctaatt tttgttgaca ctctatcgtt gatagagtta ttttaccact ccctatcagt
      721 gatagagaaa agatatacta aaagaggaga aaactgatat gatcgaaaaa aaaaagtcct
      781 tcgccaaggg catgggcgtg aagtccacac tcgtgtccgg ctccaaagtg tacatgacaa
      841 ccttcgccga aggcagcgac gccaggctgg aaaagatcgt ggagggcgac agcatcagga
      901 gcgtgaatga gggcgaggcc ttcagcgctg aaatggccga taaaaacgcc ggctataaga
      961 tcggcaacgc caaattcagc catcctaagg gctacgccgt ggtggctaac aaccctctgt
     1021 atacaggacc cgtccagcag gatatgctcg gcctgaagga aactctggaa aagaggtact
     1081 tcggcgagag cgctgatggc aatgacaata tttgtatcca ggtgatccat aacatcctgg
     1141 acattgaaaa aatcctcgcc gaatacatta ccaacgccgc ctacgccgtc aacaatatct
     1201 ccggcctgga taaggacatt attggattcg gcaagttctc cacagtgtat acctacgacg
     1261 aattcaaaga ccccgagcac catagggccg ctttcaacaa taacgataag ctcatcaacg
     1321 ccatcaaggc ccagtatgac gagttcgaca acttcctcga taaccccaga ctcggctatt
     1381 tcggccaggc ctttttcagc aaggagggca gaaattacat catcaattac ggcaacgaat
     1441 gctatgacat tctggccctc ctgagcggac tggcgcactg ggtggtcgct aacaacgaag
     1501 aagagtccag gatctccagg acctggctct acaacctcga taagaacctc gacaacgaat
     1561 acatctccac cctcaactac ctctacgaca ggatcaccaa tgagctgacc aactccttct
     1621 ccaagaactc cgccgccaac gtgaactata ttgccgaaac tctgggaatc aaccctgccg
     1681 aattcgccga acaatatttc agattcagca ttatgaaaga gcagaaaaac ctcggattca
     1741 atatcaccaa gctcagggaa gtgatgctgg acaggaagga tatgtccgag atcaggaaaa
     1801 atcataaggt gttcgactcc atcaggacca aggtctacac catgatggac tttgtgattt
     1861 ataggtatta catcgaagag gatgccaagg tggctgccgc caataagtcc ctccccgata
     1921 atgagaagtc cctgagcgag aaggatatct ttgtgattaa cctgaggggc tccttcaacg
     1981 acgaccagaa ggatgccctc tactacgatg aagctaatag aatttggaga aagctcgaaa
     2041 atatcatgca caacatcaag gaatttaggg gaaacaagac aagagagtat aagaagaagg
     2101 acgcccctag actgcccaga atcctgcccg ctggccgtga tgtttccgcc ttcagcaaac
     2161 tcatgtatgc cctgaccatg ttcctggatg gcaaggagat caacgacctc ctgaccaccc
     2221 tgattaataa attcgataac atccagagct tcctgaaggt gatgcctctc atcggagtca
     2281 acgctaagtt cgtggaggaa tacgcctttt tcaaagactc cgccaagatc gccgatgagc
     2341 tgaggctgat caagtccttc gctagaatgg gagaacctat tgccgatgcc aggagggcca
     2401 tgtatatcga cgccatccgt attttaggaa ccaacctgtc ctatgatgag ctcaaggccc
     2461 tcgccgacac cttttccctg gacgagaacg gaaacaagct caagaaaggc aagcacggca
     2521 tgagaaattt cattattaat aacgtgatca gcaataaaag gttccactac ctgatcagat
     2581 acggtgatcc tgcccacctc catgagatcg ccaaaaacga ggccgtggtg aagttcgtgc
     2641 tcggcaggat cgctgacatc cagaaaaaac agggccagaa cggcaagaac cagatcgaca
     2701 ggtactacga aacttgtatc ggaaaggata agggcaagag cgtgagcgaa aaggtggacg
     2761 ctctcacaaa gatcatcacc ggaatgaact acgaccaatt cgacaagaaa aggagcgtca
     2821 ttgaggacac cggcagggaa aacgccgaga gggagaagtt taaaaagatc atcagcctgt
     2881 acctcaccgt gatctaccac atcctcaaga atattgtcaa tatcaacgcc aggtacgtca
     2941 tcggattcca ttgcgtcgag cgtgatgctc aactgtacaa ggagaaaggc tacgacatca
     3001 atctcaagaa actggaagag aagggattca gctccgtcac caagctctgc gctggcattg
     3061 atgaaactgc ccccgataag agaaaggacg tggaaaagga gatggctgaa agagccaagg
     3121 agagcattga cagcctcgag agcgccaacc ccaagctgta tgccaattac atcaaataca
     3181 gcgacgagaa gaaagccgag gagttcacca ggcagattaa cagggagaag gccaaaaccg
     3241 ccctgaacgc ctacctgagg aacaccaagt ggaatgtgat catcagggag gacctcctga
     3301 gaattgacaa caagacatgt accctgttcg caaacaaggc cgtcgccctg gaagtggcca
     3361 ggtatgtcca cgcctatatc aacgacattg ccgaggtcaa ttcctacttc caactgtacc
     3421 attacatcat gcagagaatt atcatgaatg agaggtacga gaaaagcagc ggaaaggtgt
     3481 ccgagtactt cgacgctgtg aatgacgaga agaagtacaa cgataggctc ctgaaactgc
     3541 tgtgtgtgcc tttcggctac tgtatcccca ggtttaagaa cctgagcatc gaggccctgt
     3601 tcgataggaa cgaggccgcc aagttcgaca aggagaaaaa gaaggtgtcc ggcaattcct
     3661 aactcgagta aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     3721 ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc
     3781 ttcgggtggg cctttctgcg tttataccta gggatatatt ccgcttcctc gctcactgac
     3841 tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat
     3901 ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt
     3961 ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt
     4021 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc
     4081 gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt
     4141 ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg
     4201 cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4261 aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga
     4321 gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg
     4381 cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa
     4441 accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg
     4501 atctcaagaa gatcatctta ttaatcagat aaaatatttc tagatttcag tgcaatttat
     4561 ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgttac tagtgcttgg
     4621 attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag
     4681 ttctgaggtc attactggat ctatcaacag gagtccaagc gagctcgata tcaaattacg
     4741 ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga
     4801 agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt
     4861 gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt
     4921 ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa
     4981 taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata
     5041 tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag
     5101 tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt
     5161 ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga atgtgaataa
     5221 aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca
     5281 gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt
     5341 tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag
     5401 cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt
     5461 cattatggtg aaagttggaa cctcttacgt gccgat
//
