LOCUS       sequence-409551-        7574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing active RfxCas13d with mCherry reporter for 
            investigating CasRx activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7574)
  AUTHORS   Kumar S, Hsiao YW, Wong VHY, Aubin D, Wang JH, Lisowski L, Rakoczy 
            EP, Li F, Alarcon-Martinez L, Gonzalez-Cordero A, Bui BV, Liu GS
  TITLE     Characterization of RNA editing and gene therapy with a compact 
            CRISPR-Cas13 in the retina.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Nov 5;121(45):e2408345121. doi: 
            10.1073/pnas.2408345121. Epub 2024 Oct 30.
  PUBMED    39475642
REFERENCE   2  (bases 1 to 7574)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-11-5"; volume: "121(45):e2408345121. 
            doi"; pages: " 10.1073/pnas.2408345121. Epub 2024 Oct 30"
COMMENT     Sequence Label: pC129: pAAV.CMV-CasRx mCh
FEATURES             Location/Qualifiers
     source          1..7574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        229..532
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        533..736
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     687..707
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    757..781
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             794..814
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             824..3721
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             3731..3751
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3761..3787
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3803..3856
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3857..4567
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4002..4020)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4270..4289
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(4568..4592)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4616..4633)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4622..4829
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4837..4977
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4837..4966
                     /label=AAV2 ITR
     rep_origin      5052..5507
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5139..5158)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5349..5370
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5524..5543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5643..5665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5703..5721)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5789..5893
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5894..5962,5963..6754)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6112..6131)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6925..7513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7414..7433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgta gttattaata
      181 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      241 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      301 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      361 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      421 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      481 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
      541 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
      601 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
      661 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
      721 ctatataagc agagctggtt tagtgaaccg tcagatccaa gtttgtacaa aaaagcaggc
      781 tgccaccatg agccccaaga agaagagaaa ggtggaggcc agcatcgaaa aaaaaaagtc
      841 cttcgccaag ggcatgggcg tgaagtccac actcgtgtcc ggctccaaag tgtacatgac
      901 aaccttcgcc gaaggcagcg acgccaggct ggaaaagatc gtggagggcg acagcatcag
      961 gagcgtgaat gagggcgagg ccttcagcgc tgaaatggcc gataaaaacg ccggctataa
     1021 gatcggcaac gccaaattca gccatcctaa gggctacgcc gtggtggcta acaaccctct
     1081 gtatacagga cccgtccagc aggatatgct cggcctgaag gaaactctgg aaaagaggta
     1141 cttcggcgag agcgctgatg gcaatgacaa tatttgtatc caggtgatcc ataacatcct
     1201 ggacattgaa aaaatcctcg ccgaatacat taccaacgcc gcctacgccg tcaacaatat
     1261 ctccggcctg gataaggaca ttattggatt cggcaagttc tccacagtgt atacctacga
     1321 cgaattcaaa gaccccgagc accatagggc cgctttcaac aataacgata agctcatcaa
     1381 cgccatcaag gcccagtatg acgagttcga caacttcctc gataacccca gactcggcta
     1441 tttcggccag gcctttttca gcaaggaggg cagaaattac atcatcaatt acggcaacga
     1501 atgctatgac attctggccc tcctgagcgg actgaggcac tgggtggtcc ataacaacga
     1561 agaagagtcc aggatctcca ggacctggct ctacaacctc gataagaacc tcgacaacga
     1621 atacatctcc accctcaact acctctacga caggatcacc aatgagctga ccaactcctt
     1681 ctccaagaac tccgccgcca acgtgaacta tattgccgaa actctgggaa tcaaccctgc
     1741 cgaattcgcc gaacaatatt tcagattcag cattatgaaa gagcagaaaa acctcggatt
     1801 caatatcacc aagctcaggg aagtgatgct ggacaggaag gatatgtccg agatcaggaa
     1861 aaatcataag gtgttcgact ccatcaggac caaggtctac accatgatgg actttgtgat
     1921 ttataggtat tacatcgaag aggatgccaa ggtggctgcc gccaataagt ccctccccga
     1981 taatgagaag tccctgagcg agaaggatat ctttgtgatt aacctgaggg gctccttcaa
     2041 cgacgaccag aaggatgccc tctactacga tgaagctaat agaatttgga gaaagctcga
     2101 aaatatcatg cacaacatca aggaatttag gggaaacaag acaagagagt ataagaagaa
     2161 ggacgcccct agactgccca gaatcctgcc cgctggccgt gatgtttccg ccttcagcaa
     2221 actcatgtat gccctgacca tgttcctgga tggcaaggag atcaacgacc tcctgaccac
     2281 cctgattaat aaattcgata acatccagag cttcctgaag gtgatgcctc tcatcggagt
     2341 caacgctaag ttcgtggagg aatacgcctt tttcaaagac tccgccaaga tcgccgatga
     2401 gctgaggctg atcaagtcct tcgctagaat gggagaacct attgccgatg ccaggagggc
     2461 catgtatatc gacgccatcc gtattttagg aaccaacctg tcctatgatg agctcaaggc
     2521 cctcgccgac accttttccc tggacgagaa cggaaacaag ctcaagaaag gcaagcacgg
     2581 catgagaaat ttcattatta ataacgtgat cagcaataaa aggttccact acctgatcag
     2641 atacggtgat cctgcccacc tccatgagat cgccaaaaac gaggccgtgg tgaagttcgt
     2701 gctcggcagg atcgctgaca tccagaaaaa acagggccag aacggcaaga accagatcga
     2761 caggtactac gaaacttgta tcggaaagga taagggcaag agcgtgagcg aaaaggtgga
     2821 cgctctcaca aagatcatca ccggaatgaa ctacgaccaa ttcgacaaga aaaggagcgt
     2881 cattgaggac accggcaggg aaaacgccga gagggagaag tttaaaaaga tcatcagcct
     2941 gtacctcacc gtgatctacc acatcctcaa gaatattgtc aatatcaacg ccaggtacgt
     3001 catcggattc cattgcgtcg agcgtgatgc tcaactgtac aaggagaaag gctacgacat
     3061 caatctcaag aaactggaag agaagggatt cagctccgtc accaagctct gcgctggcat
     3121 tgatgaaact gcccccgata agagaaagga cgtggaaaag gagatggctg aaagagccaa
     3181 ggagagcatt gacagcctcg agagcgccaa ccccaagctg tatgccaatt acatcaaata
     3241 cagcgacgag aagaaagccg aggagttcac caggcagatt aacagggaga aggccaaaac
     3301 cgccctgaac gcctacctga ggaacaccaa gtggaatgtg atcatcaggg aggacctcct
     3361 gagaattgac aacaagacat gtaccctgtt cagaaacaag gccgtccacc tggaagtggc
     3421 caggtatgtc cacgcctata tcaacgacat tgccgaggtc aattcctact tccaactgta
     3481 ccattacatc atgcagagaa ttatcatgaa tgagaggtac gagaaaagca gcggaaaggt
     3541 gtccgagtac ttcgacgctg tgaatgacga gaagaagtac aacgataggc tcctgaaact
     3601 gctgtgtgtg cctttcggct actgtatccc caggtttaag aacctgagca tcgaggccct
     3661 gttcgatagg aacgaggccg ccaagttcga caaggagaaa aagaaggtgt ccggcaattc
     3721 cggatccgga cctaagaaaa agaggaaggt ggcggccgct tacccatacg atgttccaga
     3781 ttacgctgct agcggcagtg gagagggcag aggaagtctg ctaacatgcg gtgacgtcga
     3841 ggagaatcct ggcccaatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
     3901 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     3961 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     4021 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     4081 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     4141 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     4201 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     4261 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     4321 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     4381 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     4441 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     4501 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     4561 caagtaaacc cagctttctt gtacaaagtg ggaattccta gagctcgctg atcagcctcg
     4621 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     4681 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     4741 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     4801 tgggaagaga atagcaggca tgctggggag ggccgcagga acccctagtg atggagttgg
     4861 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     4921 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     4981 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     5041 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     5101 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc
     5161 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5221 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     5281 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5341 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc
     5401 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta
     5461 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     5521 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     5581 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5641 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     5701 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     5761 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5821 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5881 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5941 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     6001 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     6061 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     6121 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     6181 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     6241 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6301 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6361 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6421 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6481 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6541 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6601 tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6661 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6721 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6781 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6841 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6901 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6961 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     7021 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     7081 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     7141 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     7201 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     7261 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     7321 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     7381 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     7441 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     7501 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7561 ttttgctcac atgt
//
