LOCUS       sequence-448802-        5653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dRxCas13d expressed from J23107 promoter with J23119-driven crRNA 
            (non-cognate DR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5653)
  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 5653)
  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: pRC065d
FEATURES             Location/Qualifiers
     source          1..5653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(22..648)
                     /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     553..572
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        664..719
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    700..718
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             731..742
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             749..3649
                     /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      3676..3747
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3763..3790
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(3818..3835)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3952..4497)
                     /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(4032..4051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4605..4639
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     terminator      complement(4768..4862)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4883..5542)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5457..5476
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5543..5645)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 ttttcgccag atatcgacgt cttaagaccc actttcacat ttaagttgtt tttctaatcc
       61 gcatatgatc aattcaaggc cgaataagaa ggctggctct gcaccttggt gatcaaataa
      121 ttcgatagct tgtcgtaata atggcggcat actatcagta gtaggtgttt ccctttcttc
      181 tttagcgact tgatgctctt gatcttccaa tacgcaacct aaagtaaaat gccccacagc
      241 gctgagtgca tataatgcat tctctagtga aaaaccttgt tggcataaaa aggctaattg
      301 attttcgaga gtttcatact gtttttctgt aggccgtgta cctaaatgta cttttgctcc
      361 atcgcgatga cttagtaaag cacatctaaa acttttagcg ttattacgta aaaaatcttg
      421 ccagctttcc ccttctaaag ggcaaaagtg agtatggtgc ctatctaaca tctcaatggc
      481 taaggcgtcg agcaaagccc gcttattttt tacatgccaa tacaatgtag gctgctctac
      541 acctagcttc tgggcgagtt tacgggttgt taaaccttcg attccgacct cattaagcag
      601 ctctaatgcg ctgttaatca ctttactttt atctaatcta gacatcatta attcctaatt
      661 tttgttgaca ctctatcgtt gatagagtta ttttaccact ccctatcagt gatagagaaa
      721 agatatacta aaagaggaga aaactgatat gatcgaaaaa aaaaagtcct tcgccaaggg
      781 catgggcgtg aagtccacac tcgtgtccgg ctccaaagtg tacatgacaa ccttcgccga
      841 aggcagcgac gccaggctgg aaaagatcgt ggagggcgac agcatcagga gcgtgaatga
      901 gggcgaggcc ttcagcgctg aaatggccga taaaaacgcc ggctataaga tcggcaacgc
      961 caaattcagc catcctaagg gctacgccgt ggtggctaac aaccctctgt atacaggacc
     1021 cgtccagcag gatatgctcg gcctgaagga aactctggaa aagaggtact tcggcgagag
     1081 cgctgatggc aatgacaata tttgtatcca ggtgatccat aacatcctgg acattgaaaa
     1141 aatcctcgcc gaatacatta ccaacgccgc ctacgccgtc aacaatatct ccggcctgga
     1201 taaggacatt attggattcg gcaagttctc cacagtgtat acctacgacg aattcaaaga
     1261 ccccgagcac catagggccg ctttcaacaa taacgataag ctcatcaacg ccatcaaggc
     1321 ccagtatgac gagttcgaca acttcctcga taaccccaga ctcggctatt tcggccaggc
     1381 ctttttcagc aaggagggca gaaattacat catcaattac ggcaacgaat gctatgacat
     1441 tctggccctc ctgagcggac tggcgcactg ggtggtcgct aacaacgaag aagagtccag
     1501 gatctccagg acctggctct acaacctcga taagaacctc gacaacgaat acatctccac
     1561 cctcaactac ctctacgaca ggatcaccaa tgagctgacc aactccttct ccaagaactc
     1621 cgccgccaac gtgaactata ttgccgaaac tctgggaatc aaccctgccg aattcgccga
     1681 acaatatttc agattcagca ttatgaaaga gcagaaaaac ctcggattca atatcaccaa
     1741 gctcagggaa gtgatgctgg acaggaagga tatgtccgag atcaggaaaa atcataaggt
     1801 gttcgactcc atcaggacca aggtctacac catgatggac tttgtgattt ataggtatta
     1861 catcgaagag gatgccaagg tggctgccgc caataagtcc ctccccgata atgagaagtc
     1921 cctgagcgag aaggatatct ttgtgattaa cctgaggggc tccttcaacg acgaccagaa
     1981 ggatgccctc tactacgatg aagctaatag aatttggaga aagctcgaaa atatcatgca
     2041 caacatcaag gaatttaggg gaaacaagac aagagagtat aagaagaagg acgcccctag
     2101 actgcccaga atcctgcccg ctggccgtga tgtttccgcc ttcagcaaac tcatgtatgc
     2161 cctgaccatg ttcctggatg gcaaggagat caacgacctc ctgaccaccc tgattaataa
     2221 attcgataac atccagagct tcctgaaggt gatgcctctc atcggagtca acgctaagtt
     2281 cgtggaggaa tacgcctttt tcaaagactc cgccaagatc gccgatgagc tgaggctgat
     2341 caagtccttc gctagaatgg gagaacctat tgccgatgcc aggagggcca tgtatatcga
     2401 cgccatccgt attttaggaa ccaacctgtc ctatgatgag ctcaaggccc tcgccgacac
     2461 cttttccctg gacgagaacg gaaacaagct caagaaaggc aagcacggca tgagaaattt
     2521 cattattaat aacgtgatca gcaataaaag gttccactac ctgatcagat acggtgatcc
     2581 tgcccacctc catgagatcg ccaaaaacga ggccgtggtg aagttcgtgc tcggcaggat
     2641 cgctgacatc cagaaaaaac agggccagaa cggcaagaac cagatcgaca ggtactacga
     2701 aacttgtatc ggaaaggata agggcaagag cgtgagcgaa aaggtggacg ctctcacaaa
     2761 gatcatcacc ggaatgaact acgaccaatt cgacaagaaa aggagcgtca ttgaggacac
     2821 cggcagggaa aacgccgaga gggagaagtt taaaaagatc atcagcctgt acctcaccgt
     2881 gatctaccac atcctcaaga atattgtcaa tatcaacgcc aggtacgtca tcggattcca
     2941 ttgcgtcgag cgtgatgctc aactgtacaa ggagaaaggc tacgacatca atctcaagaa
     3001 actggaagag aagggattca gctccgtcac caagctctgc gctggcattg atgaaactgc
     3061 ccccgataag agaaaggacg tggaaaagga gatggctgaa agagccaagg agagcattga
     3121 cagcctcgag agcgccaacc ccaagctgta tgccaattac atcaaataca gcgacgagaa
     3181 gaaagccgag gagttcacca ggcagattaa cagggagaag gccaaaaccg ccctgaacgc
     3241 ctacctgagg aacaccaagt ggaatgtgat catcagggag gacctcctga gaattgacaa
     3301 caagacatgt accctgttcg caaacaaggc cgtcgccctg gaagtggcca ggtatgtcca
     3361 cgcctatatc aacgacattg ccgaggtcaa ttcctacttc caactgtacc attacatcat
     3421 gcagagaatt atcatgaatg agaggtacga gaaaagcagc ggaaaggtgt ccgagtactt
     3481 cgacgctgtg aatgacgaga agaagtacaa cgataggctc ctgaaactgc tgtgtgtgcc
     3541 tttcggctac tgtatcccca ggtttaagaa cctgagcatc gaggccctgt tcgataggaa
     3601 cgaggccgcc aagttcgaca aggagaaaaa gaaggtgtcc ggcaattcct aactcgagta
     3661 aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     3721 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     3781 cctttctgcg tttataccta gggatatatt ccgcttcctc gctcactgac tcgctacgct
     3841 cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag
     3901 atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag
     3961 gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc
     4021 gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt
     4081 tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac
     4141 gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc
     4201 ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa
     4261 gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg
     4321 aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca
     4381 agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc
     4441 aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa
     4501 gatcatctta ttaatcagat aaaatatttc tagatttcag tgcaatttat ctcttcaaat
     4561 gtagcacctg aagtcagccc catacgatat aagttgttac tagattgaca gctagctcag
     4621 tcctaggtat aatactagtc actagtgcga atttgcacta gtctaaaaca gaacgtcttc
     4681 gctactcgcc atggtacaac gcatgagaaa gcccccggaa gatcaccttc cgggggcttt
     4741 tttattgcgc cttaagctag tgcttggatt ctcaccaata aaaaacgccc ggcggcaacc
     4801 gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag
     4861 tccaagcgag ctcgatatca aattacgccc cgccctgcca ctcatcgcag tactgttgta
     4921 attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg
     4981 ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg
     5041 cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat
     5101 tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac
     5161 cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt
     5221 cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa
     5281 cactatccca tatcaccagc tcaccgtctt tcattgccat acgaaattcc ggatgagcat
     5341 tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta
     5401 cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa
     5461 ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat
     5521 atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa
     5581 aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc
     5641 gatcaacgtc tca
//
