LOCUS       sequence-463773-        7325 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a RfxCas13d-compatible gRNA and to express HA Tagged 
            hfCas13d from a 0.5 bp synapsin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7325)
  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 7325)
  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) 0.5 Syn-intron-hfCas13d-pA
FEATURES             Location/Qualifiers
     source          1..7325
                     /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)"
     intron          1704..2082
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             2165..2185
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2195..5092
                     /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             5102..5122
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5132..5158
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    5173..5221
                     /note="synthetic polyadenylation signal"
     repeat_region   5238..5378
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5238..5367
                     /label=AAV2 ITR
     rep_origin      5453..5908
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5540..5559)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5750..5771
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5925..5944)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6044..6066
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6104..6122)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6190..6294
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6295..6363,6364..7155)
                     /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(6513..6532)
                     /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 gtacaagtat ctgggaaggg taacaatgca
     1141 agttagcctg aggatttagg aggaggctga aaaacagagt aggagcctta ctacgggtcc
     1201 agaccctacg gacaagaacc cccactccca ctccccaaat tgcgcattcc ctcccccatc
     1261 agagggggag gggaagagga tgcagcgcgg cgcggcgcgt gcgcactgtc ggatttagta
     1321 ccgcggacag agccttcgcc cccgctgccg gcgcgcgcca ccacctcccc agcaccaaag
     1381 gcgggctgac gtcactctcc agccctcccc aaactcccct acctcaccgc cttggtcgcg
     1441 tccgtgcagc ggtgagtcca gtcgggccgc accacaagag gtgcaagata ggggggtgca
     1501 ggcgcgacca tacgctctgc ggcggcagag cctcagcgct gcctcagtct gcagcgggca
     1561 gcagaggagt cgcgtcgtgc cagagagcgc cgccgtgctc ctgagcccct tgcgctccgc
     1621 ccccgcggcc caccgaccca ctgccccttg gatccggccg ggaacggtgc attggaacgc
     1681 ggattccccg tgccaagagt gacgtaagta ccgcctatag agtctatagg cccacaaaaa
     1741 atgctttctt cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct
     1801 ctttctttca gggcaataat gatacaatgt atcatgcctc tttgcaccat tctaaagaat
     1861 aacagtgata atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca
     1921 tataaattgt aactgatgta agaggtttca tattgctaat agcagctaca atccagctac
     1981 cattctgctt ttattttatg gttgggataa ggctggatta ttctgagtcc aagctaggcc
     2041 cttttgctaa tcatgttcat acctcttatc ttcctcccac agctcctggg caacgtgctg
     2101 gtctgtgtgc tggcccatca ctttggcaaa gaattgggat tcgaacatcg atgccaccat
     2161 gagccccaag aagaagagaa aggtggaggc cagcatcgaa aaaaaaaagt ccttcgccaa
     2221 gggcatgggc gtgaagtcca cactcgtgtc cggctccaaa gtgtacatga caaccttcgc
     2281 cgaaggcagc gacgccaggc tggaaaagat cgtggagggc gacagcatca ggagcgtgaa
     2341 tgagggcgag gccttcagcg ctgaaatggc cgataaaaac gccggctata agatcggcaa
     2401 cgccaaattc agccatccta agggctacgc cgtggtggct aacaaccctc tgtatacagg
     2461 acccgtccag caggatatgc tcggcctgaa ggaaactctg gaaaagaggt acttcggcga
     2521 gagcgctgat ggcaatgaca atatttgtat ccaggtgatc cataacatcc tggacattga
     2581 aaaaatcctc gtggaataca ttaccaacgt ggtgtacgtg gtcaacaata tctccggcct
     2641 ggataaggac attattggat tcggcaagtt ctccacagtg tatacctacg acgaattcaa
     2701 agaccccgag caccataggg ccgctttcaa caataacgat aagctcatca acgccatcaa
     2761 ggcccagtat gacgagttcg acaacttcct cgataacccc agactcggct atttcggcca
     2821 ggcctttttc agcaaggagg gcagaaatta catcatcaat tacggcaacg aatgctatga
     2881 cattctggcc ctcctgagcg gactgaggca ctgggtggtc cataacaacg aagaagagtc
     2941 caggatctcc aggacctggc tctacaacct cgataagaac ctcgacaacg aatacatctc
     3001 caccctcaac tacctctacg acaggatcac caatgagctg accaactcct tctccaagaa
     3061 ctccgccgcc aacgtgaact atattgccga aactctggga atcaaccctg ccgaattcgc
     3121 cgaacaatat ttcagattca gcattatgaa agagcagaaa aacctcggat tcaatatcac
     3181 caagctcagg gaagtgatgc tggacaggaa ggatatgtcc gagatcagga aaaatcataa
     3241 ggtgttcgac tccatcagga ccaaggtcta caccatgatg gactttgtga tttataggta
     3301 ttacatcgaa gaggatgcca aggtggctgc cgccaataag tccctccccg ataatgagaa
     3361 gtccctgagc gagaaggata tctttgtgat taacctgagg ggctccttca acgacgacca
     3421 gaaggatgcc ctctactacg atgaagctaa tagaatttgg agaaagctcg aaaatatcat
     3481 gcacaacatc aaggaattta ggggaaacaa gacaagagag tataagaaga aggacgcccc
     3541 tagactgccc agaatcctgc ccgctggccg tgatgtttcc gccttcagca aactcatgta
     3601 tgccctgacc atgttcctgg atggcaagga gatcaacgac ctcctgacca ccctgattaa
     3661 taaattcgat aacatccaga gcttcctgaa ggtgatgcct ctcatcggag tcaacgctaa
     3721 gttcgtggag gaatacgcct ttttcaaaga ctccgccaag atcgccgatg agctgaggct
     3781 gatcaagtcc ttcgctagaa tgggagaacc tattgccgat gccaggaggg ccatgtatat
     3841 cgacgccatc cgtattttag gaaccaacct gtcctatgat gagctcaagg ccctcgccga
     3901 caccttttcc ctggacgaga acggaaacaa gctcaagaaa ggcaagcacg gcatgagaaa
     3961 tttcattatt aataacgtga tcagcaataa aaggttccac tacctgatca gatacggtga
     4021 tcctgcccac ctccatgaga tcgccaaaaa cgaggccgtg gtgaagttcg tgctcggcag
     4081 gatcgctgac atccagaaaa aacagggcca gaacggcaag aaccagatcg acaggtacta
     4141 cgaaacttgt atcggaaagg ataagggcaa gagcgtgagc gaaaaggtgg acgctctcac
     4201 aaagatcatc accggaatga actacgacca attcgacaag aaaaggagcg tcattgagga
     4261 caccggcagg gaaaacgccg agagggagaa gtttaaaaag atcatcagcc tgtacctcac
     4321 cgtgatctac cacatcctca agaatattgt caatatcaac gccaggtacg tcatcggatt
     4381 ccattgcgtc gagcgtgatg ctcaactgta caaggagaaa ggctacgaca tcaatctcaa
     4441 gaaactggaa gagaagggat tcagctccgt caccaagctc tgcgctggca ttgatgaaac
     4501 tgcccccgat aagagaaagg acgtggaaaa ggagatggct gaaagagcca aggagagcat
     4561 tgacagcctc gagagcgcca accccaagct gtatgccaat tacatcaaat acagcgacga
     4621 gaagaaagcc gaggagttca ccaggcagat taacagggag aaggccaaaa ccgccctgaa
     4681 cgcctacctg aggaacacca agtggaatgt gatcatcagg gaggacctcc tgagaattga
     4741 caacaagaca tgtaccctgt tcagaaacaa ggccgtccac ctggaagtgg ccaggtatgt
     4801 ccacgcctat atcaacgaca ttgccgaggt caattcctac ttccaactgt accattacat
     4861 catgcagaga attatcatga atgagaggta cgagaaaagc agcggaaagg tgtccgagta
     4921 cttcgacgct gtgaatgacg agaagaagta caacgatagg ctcctgaaac tgctgtgtgt
     4981 gcctttcggc tactgtatcc ccaggtttaa gaacctgagc atcgaggccc tgttcgatag
     5041 gaacgaggcc gccaagttcg acaaggagaa aaagaaggtg tccggcaatt ccggatccgg
     5101 acctaagaaa aagaggaagg tggcggccgc ttacccatac gatgttccag attacgctta
     5161 ggctaggaat tcaataaaag atctttattt tcattagatc tgtgtgttgg ttttttgtgt
     5221 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     5281 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     5341 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     5401 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     5461 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5521 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5581 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5641 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     5701 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5761 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     5821 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5881 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     5941 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     6001 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     6061 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     6121 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     6181 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6241 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6301 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6361 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6421 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6481 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6541 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6601 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6661 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6721 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6781 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6841 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6901 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6961 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     7021 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     7081 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     7141 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     7201 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     7261 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     7321 tcttc
//
