LOCUS       sequence-380380-        7419 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 7419)
  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 7419)
  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)-0.4CaMKIIa-CasRX-mCherry-pA
FEATURES             Location/Qualifiers
     source          1..7419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    257..305
                     /note="synthetic polyadenylation signal"
     repeat_region   313..453
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   313..442
                     /label=AAV2 ITR
     rep_origin      528..983
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(615..634)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     825..846
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1000..1019)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1119..1141
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1179..1197)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1265..1369
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1370..1438,1439..2230)
                     /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(1588..1607)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2401..2989
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2890..2909
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   3051..3180
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        3199..3447
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3199..3219
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3370..3389
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3449..3478
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     CDS             3899..3919
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3929..6826
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             6836..6856
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6866..6892
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6908..6961
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(6962..7419,1..250)
                     /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(7104..7122)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7372..7391
                     /label=mCherry-F
                     /note="mCherry, forward primer"
ORIGIN
        1 gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga
       61 cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct
      121 gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac
      181 catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct
      241 gtacaagtaa aagcttaata aaagatcttt attttcatta gatctgtgtg ttggtttttt
      301 gtgtgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
      361 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
      421 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
      481 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
      541 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
      601 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
      661 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
      721 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
      781 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
      841 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
      901 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
      961 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     1021 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     1081 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     1141 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     1201 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     1261 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1321 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1381 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1441 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1501 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1561 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     1621 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1681 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1741 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     1801 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     1861 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     1921 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     1981 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2041 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
     2101 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2161 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2221 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2281 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2341 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2401 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2461 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2521 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2581 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2641 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2701 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2761 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2821 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2881 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2941 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3001 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
     3061 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
     3121 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
     3181 gcggcctcta gagctagcga gggcctattt cccatgattc cttcatattt gcatatacga
     3241 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
     3301 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     3361 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     3421 tatatcttgt ggaaaggacg aaacaccgaa cccctaccaa ctggtcgggg tttgaaacgg
     3481 aagagcgagc tcttcttttt tttctgtaca acgcgtactt gtggactaag tttgttcaca
     3541 tccccttctc caaccccctc agtacatcac cctgggagaa caaggtccac ttgcttctgg
     3601 gcccacacag tcctgcagta ttgtgtatat aaggccaggg caacggagga gcaggttttg
     3661 aagtgaaagg caggcaggtg ttggggaggc agttaccggg gcaacgggaa cagggcgttt
     3721 cggaggtggt tgccatgggg acctggatgc tgacgaaggc tcgcgaggct gtgagcagcc
     3781 acagtgccct gctcagaagc cccaagctcg tcaatcaagc tggttctcca tttgcactca
     3841 ggagcacggg caggcgagtg gcccctagtt ctgggagcag accggtgcca ccatgagccc
     3901 caagaagaag agaaaggtgg aggccagcat cgaaaaaaaa aagtccttcg ccaagggcat
     3961 gggcgtgaag tccacactcg tgtccggctc caaagtgtac atgacaacct tcgccgaagg
     4021 cagcgacgcc aggctggaaa agatcgtgga gggcgacagc atcaggagcg tgaatgaggg
     4081 cgaggccttc agcgctgaaa tggccgataa aaacgccggc tataagatcg gcaacgccaa
     4141 attcagccat cctaagggct acgccgtggt ggctaacaac cctctgtata caggacccgt
     4201 ccagcaggat atgctcggcc tgaaggaaac tctggaaaag aggtacttcg gcgagagcgc
     4261 tgatggcaat gacaatattt gtatccaggt gatccataac atcctggaca ttgaaaaaat
     4321 cctcgccgaa tacattacca acgccgccta cgccgtcaac aatatctccg gcctggataa
     4381 ggacattatt ggattcggca agttctccac agtgtatacc tacgacgaat tcaaagaccc
     4441 cgagcaccat agggccgctt tcaacaataa cgataagctc atcaacgcca tcaaggccca
     4501 gtatgacgag ttcgacaact tcctcgataa ccccagactc ggctatttcg gccaggcctt
     4561 tttcagcaag gagggcagaa attacatcat caattacggc aacgaatgct atgacattct
     4621 ggccctcctg agcggactga ggcactgggt ggtccataac aacgaagaag agtccaggat
     4681 ctccaggacc tggctctaca acctcgataa gaacctcgac aacgaataca tctccaccct
     4741 caactacctc tacgacagga tcaccaatga gctgaccaac tccttctcca agaactccgc
     4801 cgccaacgtg aactatattg ccgaaactct gggaatcaac cctgccgaat tcgccgaaca
     4861 atatttcaga ttcagcatta tgaaagagca gaaaaacctc ggattcaata tcaccaagct
     4921 cagggaagtg atgctggaca ggaaggatat gtccgagatc aggaaaaatc ataaggtgtt
     4981 cgactccatc aggaccaagg tctacaccat gatggacttt gtgatttata ggtattacat
     5041 cgaagaggat gccaaggtgg ctgccgccaa taagtccctc cccgataatg agaagtccct
     5101 gagcgagaag gatatctttg tgattaacct gaggggctcc ttcaacgacg accagaagga
     5161 tgccctctac tacgatgaag ctaatagaat ttggagaaag ctcgaaaata tcatgcacaa
     5221 catcaaggaa tttaggggaa acaagacaag agagtataag aagaaggacg cccctagact
     5281 gcccagaatc ctgcccgctg gccgtgatgt ttccgccttc agcaaactca tgtatgccct
     5341 gaccatgttc ctggatggca aggagatcaa cgacctcctg accaccctga ttaataaatt
     5401 cgataacatc cagagcttcc tgaaggtgat gcctctcatc ggagtcaacg ctaagttcgt
     5461 ggaggaatac gcctttttca aagactccgc caagatcgcc gatgagctga ggctgatcaa
     5521 gtccttcgct agaatgggag aacctattgc cgatgccagg agggccatgt atatcgacgc
     5581 catccgtatt ttaggaacca acctgtccta tgatgagctc aaggccctcg ccgacacctt
     5641 ttccctggac gagaacggaa acaagctcaa gaaaggcaag cacggcatga gaaatttcat
     5701 tattaataac gtgatcagca ataaaaggtt ccactacctg atcagatacg gtgatcctgc
     5761 ccacctccat gagatcgcca aaaacgaggc cgtggtgaag ttcgtgctcg gcaggatcgc
     5821 tgacatccag aaaaaacagg gccagaacgg caagaaccag atcgacaggt actacgaaac
     5881 ttgtatcgga aaggataagg gcaagagcgt gagcgaaaag gtggacgctc tcacaaagat
     5941 catcaccgga atgaactacg accaattcga caagaaaagg agcgtcattg aggacaccgg
     6001 cagggaaaac gccgagaggg agaagtttaa aaagatcatc agcctgtacc tcaccgtgat
     6061 ctaccacatc ctcaagaata ttgtcaatat caacgccagg tacgtcatcg gattccattg
     6121 cgtcgagcgt gatgctcaac tgtacaagga gaaaggctac gacatcaatc tcaagaaact
     6181 ggaagagaag ggattcagct ccgtcaccaa gctctgcgct ggcattgatg aaactgcccc
     6241 cgataagaga aaggacgtgg aaaaggagat ggctgaaaga gccaaggaga gcattgacag
     6301 cctcgagagc gccaacccca agctgtatgc caattacatc aaatacagcg acgagaagaa
     6361 agccgaggag ttcaccaggc agattaacag ggagaaggcc aaaaccgccc tgaacgccta
     6421 cctgaggaac accaagtgga atgtgatcat cagggaggac ctcctgagaa ttgacaacaa
     6481 gacatgtacc ctgttcagaa acaaggccgt ccacctggaa gtggccaggt atgtccacgc
     6541 ctatatcaac gacattgccg aggtcaattc ctacttccaa ctgtaccatt acatcatgca
     6601 gagaattatc atgaatgaga ggtacgagaa aagcagcgga aaggtgtccg agtacttcga
     6661 cgctgtgaat gacgagaaga agtacaacga taggctcctg aaactgctgt gtgtgccttt
     6721 cggctactgt atccccaggt ttaagaacct gagcatcgag gccctgttcg ataggaacga
     6781 ggccgccaag ttcgacaagg agaaaaagaa ggtgtccggc aattccggat ccggacctaa
     6841 gaaaaagagg aaggtggccg ctgcgtaccc atacgatgtt ccagattacg ctgccagcgg
     6901 cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc
     6961 agtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     7021 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     7081 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     7141 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     7201 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     7261 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     7321 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     7381 gcagaagaag accatgggct gggaggcctc ctccgagcg
//
