LOCUS       sequence-464623-        6683 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To Express HA tagged DjCas13d-mCherry fusion protein from the 
            mammalian EFS promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6683)
  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 6683)
  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-EFS-DjCas13d-HA-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..6683
                     /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        834..1045
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1084..1104
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3754..3774
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3784..3810
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3826..4533
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3968..3986)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4236..4255
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4540..4588
                     /note="synthetic polyadenylation signal"
     repeat_region   4596..4736
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4596..4725
                     /label=AAV2 ITR
     rep_origin      4811..5266
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4898..4917)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5108..5129
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5283..5302)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5402..5424
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5462..5480)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5548..5652
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5653..5721,5722..6513)
                     /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(5871..5890)
                     /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 gcggcctcta gagctaggcg tcctagagtc gagtggctcc ggtgcccgtc agtgggcaga
      841 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaacgggtgc
      901 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
      961 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     1021 caacgggttt gccgccagaa cacaggtgtc gtgacgcgga ccggtgctag cgccaccatg
     1081 agccccaaga agaagagaaa ggtggaggcc agcatcaaga agcaaaaatc caagaaaacg
     1141 gtttctaaaa cctccggatt gaaagaggca ctttctgtgc aggggactgt cataatgacg
     1201 tcttttggga aggggaacat ggcgaatctc tcttacaaga tcccgagctc tcaaaagccc
     1261 caaaatctca atagtagcgc cggcttgaag aacgtcgaag tatctggtaa aaagattaag
     1321 ttccaaggtc gacacccgaa gatcgccact accgacaacc ccctgtttaa gccgcagcca
     1381 ggtatggacc ttctgtgcct gaaagacaaa ctcgaaatgc actattttgg gaagactttc
     1441 gacgacaata tccacataca attgatatat caaatactgg atattgaaaa gatccttgcg
     1501 gttcatgtta acaacatcgt attcacactg gacaatgtcc tgcatccgca aaaggaagag
     1561 ctgactgaag atttcattgg ggcagggggc tggcggatta atctcgatta ccaaactctt
     1621 agagggcaga ctaacaaata tgataggttt aaaaactaca ttaaaagaaa agaacttctt
     1681 tatttcgggg aggcatttta ccatgaaaac gagagacggt atgaggaaga catcttcgca
     1741 atacttaccc tgctgtctgc ccttagacag ttttgctttc attccgatct gagtagtgat
     1801 gagagcgacc acgtgaattc attctggctg tatcaactcg aggaccaact gtctgacgag
     1861 ttcaaggaga ctctcagtat tttgtgggaa gaggtcaccg aaaggataga tagtgaattc
     1921 cttaaaacta atacagtaaa cctccatatt ttgtgtcatg ttttcccgaa agagagtaag
     1981 gagacgattg tacgcgctta ttatgaattc ctgatcaaga agtcctttaa aaatatggga
     2041 tttagcatca agaaattgcg agagattatg ctcgagcaat cagatcttaa gagtttcaaa
     2101 gaggataagt acaactccgt cagggctaaa ctgtataagc tcttcgactt tattataacc
     2161 tattactacg accaccatgc ttttgagaag gaggcgctcg tgagctcctt gcgaagttca
     2221 ctcactgagg aaaacaagga agagatttac atcaaaacgg cccggacact cgcgagcgct
     2281 cttggggccg atttcaagaa agccgctgcc gatgtaaatg ccaaaaacat aagggactat
     2341 cagaagaagg cgaacgacta taggataagt tttgaagata ttaaaatagg gaacacaggc
     2401 attgggtatt tctcagagct catatacatg ctcacccttc tcctcgatgg aaaggagata
     2461 aacgacctcc tcacaaccct gatcaacaaa tttgataata taatctcctt catagacatc
     2521 ctgaagaaac tcaatttgga atttaaattc aagcctgaat acgcggactt ctttaacatg
     2581 accaactgca ggtatactct cgaagagctg cgggttatca acagtatcgc tagaatgcaa
     2641 aagccaagcg ccgacgcccg aaagatcatg tatcgcgatg ctctgcgcat attgggaatg
     2701 gacaatcggc cggatgaaga gattgatcgg gaactcgaaa ggacaatgcc cgtgggtgcg
     2761 gatggcaaat ttatcaaagg taaacagggc tttcgcaatt tcattgcttc caacgttatt
     2821 gaatcttcac gcttccatta cctcgtacgg tataataatc cacacaagac acggactctt
     2881 gtgaaaaacc ctaatgtagt aaaatttgtg ctcgaaggga ttccggagac acaaataaaa
     2941 cgatactttg atgtttgtaa aggtcaagag atcccaccga cgtctgacaa gtccgcacag
     3001 atagacgttc ttgcccgaat aattagttca gtagactata aaatatttga agacgtaccc
     3061 caaagtgcaa agatcaataa agatgatcct agccgaaact tcagtgacgc gctgaaaaaa
     3121 cagcggtacc aggcaattgt aagtctttat ttgactgtta tgtaccttat aaccaaaaac
     3181 ctggtgtatg tcaatagtag gtatgtgatt gcatttcatt gcctggaacg agacgcattc
     3241 cttcacggtg tgacgctccc taagatgaat aaaaaaattg tgtacagtca attgactact
     3301 cacctcttga ctgacaaaaa ttacactacg tacggccacc tgaagaacca gaaaggacac
     3361 cgcaaatggt acgttcttgt caaaaataac cttcagaata gcgacataac agctgtgagt
     3421 tcattccgca acatagttgc tcacatatcc gttgtgagga atagtaatga atacataagc
     3481 gggatcggag aactgcactc ttacttcgaa ctttatcact atctcgttca atcaatgatc
     3541 gcaaagaata attggtatga taccagccac cagccgaaga ccgccgaata tctgaacaat
     3601 ctgaaaaagc accacactta ttgtaaggac tttgtaaaag cgtactgtat cccgttcgga
     3661 tacgtcgtgc cgcggtacaa aaacctcacg ataaatgaac tttttgatcg aaataacccg
     3721 aatcccgaac caaaagagga agtgggatcc ggacctaaga aaaagaggaa ggtggccgct
     3781 gcgtacccat acgatgttcc agattacgct gccagcggca gtggagtgag caagggcgag
     3841 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     3901 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     3961 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     4021 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     4081 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     4141 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     4201 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     4261 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     4321 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     4381 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     4441 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     4501 tccaccggcg gcatggacga gctgtacaag taaaagctta ataaaagatc tttattttca
     4561 ttagatctgt gtgttggttt tttgtgtgcg gccgcaggaa cccctagtga tggagttggc
     4621 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4681 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     4741 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4801 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4861 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     4921 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4981 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     5041 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5101 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     5161 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     5221 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     5281 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5341 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5401 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5461 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5521 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5581 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5641 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5701 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5761 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5821 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5881 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5941 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6001 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6061 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6121 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6181 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6241 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6301 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6361 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6421 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6481 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6541 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6601 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6661 gtagaaaaga tcaaaggatc ttc
//
