LOCUS       sequence-463789-        6740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To Express HA tagged DjCas13d and mcherry from the mammalian EFS 
            promoter. The DjCas13dx and mCherry are separated by a P2A site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6740)
  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 6740)
  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-P2A-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..6740
                     /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..3882
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3883..4590
                     /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(4025..4043)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4293..4312
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4597..4645
                     /note="synthetic polyadenylation signal"
     repeat_region   4653..4793
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4653..4782
                     /label=AAV2 ITR
     rep_origin      4868..5323
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4955..4974)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5165..5186
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5340..5359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5459..5481
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5519..5537)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5605..5709
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5710..5778,5779..6570)
                     /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(5928..5947)
                     /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 gccagcggct ccggagccac gaacttctct
     3841 ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc ccgtgagcaa gggcgaggag
     3901 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     3961 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     4021 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     4081 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     4141 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     4201 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     4261 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     4321 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     4381 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     4441 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     4501 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     4561 accggcggca tggacgagct gtacaagtaa aagcttaata aaagatcttt attttcatta
     4621 gatctgtgtg ttggtttttt gtgtgcggcc gcaggaaccc ctagtgatgg agttggccac
     4681 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4741 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4801 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4861 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4921 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     4981 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5041 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     5101 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5161 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     5221 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     5281 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     5341 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     5401 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5461 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     5521 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     5581 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5641 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5701 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5761 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5821 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5881 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5941 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     6001 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     6061 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6121 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6181 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6241 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6301 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6361 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6421 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6481 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6541 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6601 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6661 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6721 gaaaagatca aaggatcttc
//
