LOCUS       sequence-401408-        6659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing active RfxCas13d with non-targeting gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6659)
  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 6659)
  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: pM148: pAAV.CMV-Cas13e-NT sgRNA
FEATURES             Location/Qualifiers
     source          1..6659
                     /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"
     promoter        169..417
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     169..189
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     340..359
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    609..633
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        694..997
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        998..1201
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1152..1172
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(1223..1247)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1256..1276
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3599..3619
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3626..3652
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(3656..3676)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    3707..3914
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3922..4062
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3922..4051
                     /label=AAV2 ITR
     rep_origin      4137..4592
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4224..4243)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4434..4455
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4609..4628)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4728..4750
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4788..4806)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4874..4978
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4979..5047,5048..5839)
                     /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(5197..5216)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6010..6598
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6499..6518
                     /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 aaaagttgga gggcctattt
      181 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
      241 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
      301 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
      361 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggc
      421 tggagcagcc cccgatttgt ggggtgatta cagctcacca gaagcgtacc atactcacga
      481 acaggctgga gcagcccccg atttgtgggg tgattacagc tttttttgaa ttctgatgcg
      541 gtgaagcttt agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      601 ggagttccgc aagtttgtac aaaaaagcag gcttagttat taatagtaat caattacggg
      661 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      721 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      781 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      841 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      901 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      961 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     1021 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     1081 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     1141 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     1201 tggtttagtg aaccgtcaga tcacccagct ttcttgtaca aagtgggcca ccatgccaaa
     1261 gaagaaacgg aaggtcgctc aggtctctaa gcagacatcc aagaagaggg aactttccat
     1321 tgatgaatat caaggtgcca gaaaatggtg ttttacaatc gcctttaata aggccttggt
     1381 caatcgggac aagaatgacg gacttttcgt tgaatccctt ctccgccacg aaaaatactc
     1441 caagcatgat tggtacgatg aggacaccag ggcattgatc aaatgtagca cacaagccgc
     1501 aaatgctaag gcagaggctc ttcgaaatta cttctcacat tacaggcaca gtccagggtg
     1561 cttgaccttc accgcagagg acgagttgcg gactattatg gagcgggcat acgaaagggc
     1621 catattcgaa tgtagacgac gcgaaacaga agtcattatt gagttcccct cactgttcga
     1681 gggcgatagg ataactacag ctggggtagt cttttttgtc tctttcttcg ttgaaagacg
     1741 agtgctcgac cgactctacg gagccgtgag cggtctgaaa aagaatgagg gccaatacaa
     1801 actcacccga aaggcactct ccatgtactg tcttaaggat tcccgattta ccaaggcatg
     1861 ggacaaaagg gtattgttgt tcagggatat cctggcacag ttggggcgaa ttcccgccga
     1921 agcatatgaa tactaccacg gcgaacaggg tgataaaaaa cgcgctaatg ataacgaggg
     1981 cacaaatcca aagcgccata aagacaaatt cattgaattc gcattgcatt atcttgaagc
     2041 ccagcacagc gaaatctgct tcggtaggcg ccacattgta cgagaagagg ctggtgctgg
     2101 agatgaacat aagaagcacc gcaccaaggg gaaggtagtt gtcgatttta gtaagaaaga
     2161 tgaagaccag tcatattaca tatccaagaa taacgtaatt gtgcgcattg ataagaacgc
     2221 cggaccacgc tcttatagaa tggggctgaa tgagctcaag tacttggtct tgcttagcct
     2281 ccagggaaaa ggggatgacg ctattgctaa gctctatcgc tatagacaac acgttgagaa
     2341 tatcctcgac gttgtgaaag ttacagataa ggataatcat gtattcctgc ctagatttgt
     2401 gttggaacag cacggcattg gtaggaaggc attcaagcaa aggatcgatg gaagagtcaa
     2461 gcatgtgcgc ggagtgtggg aaaaaaaaaa agcagccact aacgagatga ccctgcacga
     2521 aaaagctagg gatatccttc agtatgtaaa cgaaaactgt actcgctcct ttaatcctgg
     2581 agaatacaat agactgcttg tgtgccttgt tgggaaggat gtagagaact ttcaagccgg
     2641 ccttaagcga ctccagttgg ctgaacggat agatggacga gtgtatagta tattcgcaca
     2701 gacttctaca atcaatgaaa tgcatcaagt cgtttgtgac caaattttga acaggctttg
     2761 caggatcggt gatcaaaagc tctatgacta tgtcgggctg ggtaagaagg atgaaataga
     2821 ttacaaacag aaggtcgcct ggtttaagga gcacatttcc atcagacggg ggtttttgcg
     2881 gaagaaattt tggtacgatt ctaaaaaagg atttgcaaag ctggtggaag aacatttgga
     2941 gagcggaggc gggcagcgcg acgtaggtct tgataagaaa tactaccaca tagacgcaat
     3001 tgggcggttc gagggagcaa accctgcctt gtatgaaacc ctcgcccgag atagattgtg
     3061 cttgatgatg gcacaatatt ttctgggttc agtacggaag gaacttggta acaagattgt
     3121 ttggagcaat gacagcatcg aattgcctgt tgaagggagt gtcggtaatg aaaagtccat
     3181 tgtattttcc gtgagtgact atggcaagtt gtatgttctt gacgatgcag aattccttgg
     3241 gcgaatctgc gagtacttca tgccccacga aaagggtaaa atacgctatc atacagttta
     3301 tgagaaaggc tttcgagcat ataatgacct gcaaaagaag tgtgtcgaag cagtccttgc
     3361 tttcgaagaa aaggtcgtaa aagcaaagaa gatgtccgaa aaagagggcg cccattacat
     3421 tgacttcaga gagatccttg cccagacaat gtgtaaggaa gccgagaaaa ctgctgtgaa
     3481 taaggtaagg agagcctttt tccatcacca cctcaagttc gtcatagacg aatttggatt
     3541 gttttctgat gtgatgaaaa aatatgggat tgagaaagaa tggaagtttc cagtgaagcc
     3601 caagaaaaaa agaaaagtat cctcctaccc atacgatgtt ccagattacg cttgacaact
     3661 ttattataca tagttggaat tcctagagct cgctgatcag cctcgactgt gccttctagt
     3721 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3781 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3841 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc
     3901 aggcatgctg gggagggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     3961 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4021 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     4081 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     4141 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4201 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4261 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4321 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4381 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4441 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     4501 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4561 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4621 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4681 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4741 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4801 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4861 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4921 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4981 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5041 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5101 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5161 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5221 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5281 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5341 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5401 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5461 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5521 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5581 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5641 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg
     5701 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5761 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5821 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5881 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5941 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6001 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6061 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6121 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6181 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6241 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6301 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6361 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6421 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6481 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6541 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6601 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
