LOCUS       sequence-380382-        5945 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express CasRX and a gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5945)
  AUTHORS   Back F, Sandoval A, Vu LM, Hong VM, Bhaskara A, Rodriguez SR, 
            O'Brien JT, Kolber BJ, Kroener S, Ploski JE
  TITLE     Adeno-associated viral vector resource for the RNA-targeting Cas13d:
            A comparison of high-fidelity variants, DjCas13d and hfCas13d.
  JOURNAL   Mol Ther Methods Clin Dev. 2025 Aug 20;33(4):101565. doi: 
            10.1016/j.omtm.2025.101565. eCollection 2025 Dec 11.
  PUBMED    41020266
REFERENCE   2  (bases 1 to 5945)
  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: "Mol Ther 
            Methods Clin Dev."; date: "2025-08-20"; volume: "33(4):101565. doi";
            pages: " 10.1016/j.omtm.2025.101565. eCollection 2025 Dec 11"
COMMENT     Sequence Label: pAAV-U6-DR30(SapI)-CMV-intron-GFP-pA
FEATURES             Location/Qualifiers
     source          1..5945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   11..140
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        155..403
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     155..175
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     326..345
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        405..434
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     enhancer        534..837
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        838..1041
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     992..1012
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1038..1062
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1190..1568
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     regulatory      1668..1677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1674..1676,1677..1679,1680..2393)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1674..1676,1677..1679,1680..2390)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1719..1740)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1980..1999)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2327..2348
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2446..2922
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2645..2664)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     polyA_signal    2970..3104
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2994..3013)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3048..3067
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3218..3358
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3218..3347
                     /label=AAV2 ITR
     rep_origin      3433..3888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3520..3539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3730..3751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3905..3924)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4024..4046
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4084..4102)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4170..4274
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4275..4343,4344..5135)
                     /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(4493..4512)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5306..5894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5795..5814
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctcacatgt cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg
       61 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa
      121 ctccatcact aggggttcct gcggccgcac tagcgagggc ctatttccca tgattccttc
      181 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
      241 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
      301 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
      361 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgaacccc taccaactgg
      421 tcggggtttg aaacggaaga gcgagctctt cttttttttc tgtacaacgc gtctagttat
      481 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      541 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      601 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      661 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      721 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      781 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      841 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      901 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      961 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
     1021 gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca
     1081 cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccgcgg attcgaatcc
     1141 cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc
     1201 ctatagagtc tataggccca caaaaaatgc tttcttcttt taatatactt ttttgtttat
     1261 cttatttcta atactttccc taatctcttt ctttcagggc aataatgata caatgtatca
     1321 tgcctctttg caccattcta aagaataaca gtgataattt ctgggttaag gcaatagcaa
     1381 tatttctgca tataaatatt tctgcatata aattgtaact gatgtaagag gtttcatatt
     1441 gctaatagca gctacaatcc agctaccatt ctgcttttat tttatggttg ggataaggct
     1501 ggattattct gagtccaagc taggcccttt tgctaatcat gttcatacct cttatcttcc
     1561 tcccacagct cctgggcaac gtgctggtct gtgtgctggc ccatcacttt ggcaaagaat
     1621 tgggattcga acatcgattg aattctgcag tcggggatcc accggtcgcc accatggtga
     1681 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1741 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     1801 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1861 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     1921 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1981 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     2041 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     2101 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     2161 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     2221 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2281 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2341 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc
     2401 gacctgcaga agcttgcctc gagcagcgct gctcgagaga tctacgggtg gcatccctgt
     2461 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     2521 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     2581 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     2641 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     2701 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     2761 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     2821 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     2881 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     2941 gggcccaatg gcgcgctaga taatacgtag atccagacat gataagatac attgatgagt
     3001 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     3061 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     3121 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaattc ttgcggccgc
     3181 ggcggtaccg gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     3241 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3301 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     3361 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3421 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3481 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     3541 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3601 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     3661 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3721 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     3781 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     3841 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     3901 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     3961 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4021 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4081 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4141 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4201 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4261 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4321 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4381 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4441 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4501 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4561 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4621 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4681 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4741 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4801 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4861 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4921 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     4981 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5041 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5101 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5161 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5221 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5281 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5341 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5401 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5461 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5521 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5581 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5641 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5701 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5761 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5821 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5881 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5941 ctttt
//
