LOCUS       sequence-380389-        5951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express DjCas13d compatible gRNA and GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5951)
  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 5951)
  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-GFP-pA
FEATURES             Location/Qualifiers
     source          1..5951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      18..606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     507..526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   668..797
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        812..1060
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     812..832
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     983..1002
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1197..1500
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1501..1704
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1655..1675
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1701..1725
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1853..2231
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     regulatory      2331..2340
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2337..2339,2340..2342,2343..3056)
                     /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(2337..2339,2340..2342,2343..3053)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2382..2403)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2643..2662)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2990..3011
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3109..3585
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3308..3327)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     polyA_signal    3633..3767
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3657..3676)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3711..3730
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3881..4021
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3881..4010
                     /label=AAV2 ITR
     rep_origin      4096..4551
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4183..4202)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4393..4414
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4568..4587)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4687..4709
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4747..4765)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4833..4937
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4938..5006,5007..5798)
                     /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(5156..5175)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
       61 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
      121 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
      181 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
      241 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
      301 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
      361 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
      421 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      481 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      541 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      601 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      661 cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc
      721 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
      781 catcactagg ggttcctgcg gccgcacgcg tgagggccta tttcccatga ttccttcata
      841 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
      901 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      961 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     1021 tttcttggct ttatatatct tgtggaaagg acgaaacacc gcaactacaa ccccgtaaaa
     1081 atacggggtt ctgaaacgga agagcgagct cttctttttt ttctgtacaa cgcgtctagt
     1141 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     1201 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     1261 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     1321 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     1381 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     1441 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     1501 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     1561 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     1621 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     1681 tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat
     1741 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg cggattcgaa
     1801 tcccggccgg gaacggtgca ttggaacgcg gattccccgt gccaagagtg acgtaagtac
     1861 cgcctataga gtctataggc ccacaaaaaa tgctttcttc ttttaatata cttttttgtt
     1921 tatcttattt ctaatacttt ccctaatctc tttctttcag ggcaataatg atacaatgta
     1981 tcatgcctct ttgcaccatt ctaaagaata acagtgataa tttctgggtt aaggcaatag
     2041 caatatttct gcatataaat atttctgcat ataaattgta actgatgtaa gaggtttcat
     2101 attgctaata gcagctacaa tccagctacc attctgcttt tattttatgg ttgggataag
     2161 gctggattat tctgagtcca agctaggccc ttttgctaat catgttcata cctcttatct
     2221 tcctcccaca gctcctgggc aacgtgctgg tctgtgtgct ggcccatcac tttggcaaag
     2281 aattgggatt cgaacatcga ttgaattctg cagtcgggga tccaccggtc gccaccatgg
     2341 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2401 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2461 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2521 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2581 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2641 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2701 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2761 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2821 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2881 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2941 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     3001 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagcg
     3061 gccgacctgc agaagcttgc ctcgagcagc gctgctcgag agatctacgg gtggcatccc
     3121 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc
     3181 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt
     3241 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc
     3301 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc
     3361 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag
     3421 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata
     3481 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa
     3541 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta
     3601 accgggccca atggcgcgct agataatacg tagatccaga catgataaga tacattgatg
     3661 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     3721 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     3781 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcttgcggc
     3841 cgcggcggta ccggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     3901 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3961 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     4021 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     4081 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4141 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     4201 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4261 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     4321 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4381 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     4441 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     4501 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     4561 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     4621 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4681 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     4741 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     4801 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4861 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4921 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4981 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5041 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5101 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5161 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5221 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5281 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5341 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5401 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5461 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5521 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5581 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5641 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5701 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5761 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5821 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5881 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5941 accccgtaga a
//
