LOCUS       sequence-463793-        5492 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a DjCas13d-compatible gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5492)
  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 5492)
  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-DR36(DjCas13d-SapI)-CMV-intron-MCS-pA
FEATURES             Location/Qualifiers
     source          1..5492
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        795..1043
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        1119..1367
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     enhancer        1504..1807
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1808..2011
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1962..1982
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2008..2032
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          2160..2538
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     polyA_signal    2633..3109
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2832..2851)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     polyA_signal    3157..3291
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3181..3200)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3235..3254
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3405..3545
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3405..3534
                     /label=AAV2 ITR
     rep_origin      3620..4075
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3707..3726)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3917..3938
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4092..4111)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4211..4233
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4271..4289)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4357..4461
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4462..4530,4531..5322)
                     /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(4680..4699)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat atacgataca
      841 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
      901 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      961 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     1021 tcttgtggaa aggacgaaac accgcaacta caaccccgta aaaatacggg gttctgaaac
     1081 ggaagagcga gctcttcttt tttttctgta caacgcgtga gggcctattt cccatgattc
     1141 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     1201 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     1261 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     1321 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgca actacaaccc
     1381 cgtaaaaata cggggttctg aaacggaaga gcgagctctt cttttttttc tgtacaacgc
     1441 gtctagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     1501 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     1561 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     1621 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     1681 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     1741 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     1801 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     1861 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     1921 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     1981 tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag
     2041 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccgcgg
     2101 attcgaatcc cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
     2161 taagtaccgc ctatagagtc tataggccca caaaaaatgc tttcttcttt taatatactt
     2221 ttttgtttat cttatttcta atactttccc taatctcttt ctttcagggc aataatgata
     2281 caatgtatca tgcctctttg caccattcta aagaataaca gtgataattt ctgggttaag
     2341 gcaatagcaa tatttctgca tataaatatt tctgcatata aattgtaact gatgtaagag
     2401 gtttcatatt gctaatagca gctacaatcc agctaccatt ctgcttttat tttatggttg
     2461 ggataaggct ggattattct gagtccaagc taggcccttt tgctaatcat gttcatacct
     2521 cttatcttcc tcccacagct cctgggcaac gtgctggtct gtgtgctggc ccatcacttt
     2581 ggcaaagaat tgggattcga acatcgattg aattctgcag tcggggatct acgggtggca
     2641 tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac
     2701 cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa
     2761 tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag
     2821 ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc
     2881 aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt
     2941 ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac
     3001 catattggcc aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc
     3061 caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgattttgta
     3121 ggtaaccggg cccaatggcg cgctagataa tacgtagatc cagacatgat aagatacatt
     3181 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     3241 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     3301 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattcttg
     3361 cggccgcggc ggtaccggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     3421 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     3481 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     3541 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3601 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3661 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     3721 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3781 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     3841 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3901 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     3961 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     4021 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     4081 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     4141 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4201 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4261 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     4321 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4381 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4441 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4501 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4561 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     4621 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     4681 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     4741 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     4801 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     4861 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     4921 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     4981 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5041 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5101 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5161 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5221 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5281 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5341 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5401 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5461 tcagaccccg tagaaaagat caaaggatct tc
//
