LOCUS       sequence-465997-        7175 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a RfxCas13d-compatible gRNA and to express HA Tagged 
            hfCas13d from a CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7175)
  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 7175)
  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-DR(RfxCas13d)-CAG-hfCas13d-pa
FEATURES             Location/Qualifiers
     source          1..7175
                     /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)"
     primer_bind     795..815
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     966..985
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1045..1074
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     enhancer        1174..1477
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1479..1756
                     /label=chicken beta-actin promoter
     intron          1757..1984
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             2015..2035
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2045..4942
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILVEYITNVVYVVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             4952..4972
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4982..5008
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5023..5071
                     /note="synthetic polyadenylation signal"
     repeat_region   5088..5228
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5088..5217
                     /label=AAV2 ITR
     rep_origin      5303..5758
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5390..5409)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5600..5621
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5775..5794)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5894..5916
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5954..5972)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6040..6144
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6145..6213,6214..7005)
                     /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(6363..6382)
                     /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 tagcgagggc 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 accgaacccc taccaactgg tcggggtttg aaacggaaga
     1081 gcgagctctt cttttttttc tgtacaacgc gtctagttat taatagtaat caattacggg
     1141 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     1201 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     1261 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     1321 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     1381 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     1441 gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg
     1501 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
     1561 attattttgt gcagcgatgg gggcgggggg gggggggggg tgcgcgccag gggggggggg
     1621 ggggggggag ggggggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
     1681 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
     1741 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
     1801 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     1861 ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg
     1921 gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt
     1981 tcaggttgga ccggtcatcg atgccaccat gagccccaag aagaagagaa aggtggaggc
     2041 cagcatcgaa aaaaaaaagt ccttcgccaa gggcatgggc gtgaagtcca cactcgtgtc
     2101 cggctccaaa gtgtacatga caaccttcgc cgaaggcagc gacgccaggc tggaaaagat
     2161 cgtggagggc gacagcatca ggagcgtgaa tgagggcgag gccttcagcg ctgaaatggc
     2221 cgataaaaac gccggctata agatcggcaa cgccaaattc agccatccta agggctacgc
     2281 cgtggtggct aacaaccctc tgtatacagg acccgtccag caggatatgc tcggcctgaa
     2341 ggaaactctg gaaaagaggt acttcggcga gagcgctgat ggcaatgaca atatttgtat
     2401 ccaggtgatc cataacatcc tggacattga aaaaatcctc gtggaataca ttaccaacgt
     2461 ggtgtacgtg gtcaacaata tctccggcct ggataaggac attattggat tcggcaagtt
     2521 ctccacagtg tatacctacg acgaattcaa agaccccgag caccataggg ccgctttcaa
     2581 caataacgat aagctcatca acgccatcaa ggcccagtat gacgagttcg acaacttcct
     2641 cgataacccc agactcggct atttcggcca ggcctttttc agcaaggagg gcagaaatta
     2701 catcatcaat tacggcaacg aatgctatga cattctggcc ctcctgagcg gactgaggca
     2761 ctgggtggtc cataacaacg aagaagagtc caggatctcc aggacctggc tctacaacct
     2821 cgataagaac ctcgacaacg aatacatctc caccctcaac tacctctacg acaggatcac
     2881 caatgagctg accaactcct tctccaagaa ctccgccgcc aacgtgaact atattgccga
     2941 aactctggga atcaaccctg ccgaattcgc cgaacaatat ttcagattca gcattatgaa
     3001 agagcagaaa aacctcggat tcaatatcac caagctcagg gaagtgatgc tggacaggaa
     3061 ggatatgtcc gagatcagga aaaatcataa ggtgttcgac tccatcagga ccaaggtcta
     3121 caccatgatg gactttgtga tttataggta ttacatcgaa gaggatgcca aggtggctgc
     3181 cgccaataag tccctccccg ataatgagaa gtccctgagc gagaaggata tctttgtgat
     3241 taacctgagg ggctccttca acgacgacca gaaggatgcc ctctactacg atgaagctaa
     3301 tagaatttgg agaaagctcg aaaatatcat gcacaacatc aaggaattta ggggaaacaa
     3361 gacaagagag tataagaaga aggacgcccc tagactgccc agaatcctgc ccgctggccg
     3421 tgatgtttcc gccttcagca aactcatgta tgccctgacc atgttcctgg atggcaagga
     3481 gatcaacgac ctcctgacca ccctgattaa taaattcgat aacatccaga gcttcctgaa
     3541 ggtgatgcct ctcatcggag tcaacgctaa gttcgtggag gaatacgcct ttttcaaaga
     3601 ctccgccaag atcgccgatg agctgaggct gatcaagtcc ttcgctagaa tgggagaacc
     3661 tattgccgat gccaggaggg ccatgtatat cgacgccatc cgtattttag gaaccaacct
     3721 gtcctatgat gagctcaagg ccctcgccga caccttttcc ctggacgaga acggaaacaa
     3781 gctcaagaaa ggcaagcacg gcatgagaaa tttcattatt aataacgtga tcagcaataa
     3841 aaggttccac tacctgatca gatacggtga tcctgcccac ctccatgaga tcgccaaaaa
     3901 cgaggccgtg gtgaagttcg tgctcggcag gatcgctgac atccagaaaa aacagggcca
     3961 gaacggcaag aaccagatcg acaggtacta cgaaacttgt atcggaaagg ataagggcaa
     4021 gagcgtgagc gaaaaggtgg acgctctcac aaagatcatc accggaatga actacgacca
     4081 attcgacaag aaaaggagcg tcattgagga caccggcagg gaaaacgccg agagggagaa
     4141 gtttaaaaag atcatcagcc tgtacctcac cgtgatctac cacatcctca agaatattgt
     4201 caatatcaac gccaggtacg tcatcggatt ccattgcgtc gagcgtgatg ctcaactgta
     4261 caaggagaaa ggctacgaca tcaatctcaa gaaactggaa gagaagggat tcagctccgt
     4321 caccaagctc tgcgctggca ttgatgaaac tgcccccgat aagagaaagg acgtggaaaa
     4381 ggagatggct gaaagagcca aggagagcat tgacagcctc gagagcgcca accccaagct
     4441 gtatgccaat tacatcaaat acagcgacga gaagaaagcc gaggagttca ccaggcagat
     4501 taacagggag aaggccaaaa ccgccctgaa cgcctacctg aggaacacca agtggaatgt
     4561 gatcatcagg gaggacctcc tgagaattga caacaagaca tgtaccctgt tcagaaacaa
     4621 ggccgtccac ctggaagtgg ccaggtatgt ccacgcctat atcaacgaca ttgccgaggt
     4681 caattcctac ttccaactgt accattacat catgcagaga attatcatga atgagaggta
     4741 cgagaaaagc agcggaaagg tgtccgagta cttcgacgct gtgaatgacg agaagaagta
     4801 caacgatagg ctcctgaaac tgctgtgtgt gcctttcggc tactgtatcc ccaggtttaa
     4861 gaacctgagc atcgaggccc tgttcgatag gaacgaggcc gccaagttcg acaaggagaa
     4921 aaagaaggtg tccggcaatt ccggatccgg acctaagaaa aagaggaagg tggcggccgc
     4981 ttacccatac gatgttccag attacgctta ggctaggaat tcaataaaag atctttattt
     5041 tcattagatc tgtgtgttgg ttttttgtgt gcggaccgag cggccgcagg aacccctagt
     5101 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     5161 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     5221 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     5281 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5341 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     5401 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5461 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     5521 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5581 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     5641 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     5701 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     5761 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     5821 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5881 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5941 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     6001 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6061 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6121 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     6181 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6241 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6301 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6361 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6421 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6481 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6541 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6601 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6661 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6721 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6781 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6841 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6901 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6961 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     7021 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     7081 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     7141 gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
