LOCUS       sequence-401372-        7199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing active RfxCas13d with VEGFA mRNA targeting gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7199)
  AUTHORS   Kumar S, Hsiao YW, Wong VHY, Aubin D, Wang JH, Lisowski L, Rakoczy 
            EP, Li F, Alarcon-Martinez L, Gonzalez-Cordero A, Bui BV, Liu GS
  TITLE     Characterization of RNA editing and gene therapy with a compact 
            CRISPR-Cas13 in the retina.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Nov 5;121(45):e2408345121. doi: 
            10.1073/pnas.2408345121. Epub 2024 Oct 30.
  PUBMED    39475642
REFERENCE   2  (bases 1 to 7199)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-11-5"; volume: "121(45):e2408345121. 
            doi"; pages: " 10.1073/pnas.2408345121. Epub 2024 Oct 30"
COMMENT     Sequence Label: pC130: pAAV.CMV-CasRx-VEGFA sgRNA
FEATURES             Location/Qualifiers
     source          1..7199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        169..417
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     169..189
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     340..359
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        419..454
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        485..520
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     protein_bind    549..573
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        634..937
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        938..1141
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1092..1112
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(1163..1187)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1199..1219
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1229..4126
                     /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             4136..4156
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4166..4192
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(4196..4216)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(4241..4258)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4247..4454
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4462..4602
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4462..4591
                     /label=AAV2 ITR
     rep_origin      4677..5132
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4764..4783)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4974..4995
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5149..5168)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5268..5290
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5328..5346)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5414..5518
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5519..5587,5588..6379)
                     /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(5737..5756)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6550..7138
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7039..7058
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgga gggcctattt
      181 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
      241 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
      301 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
      361 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgca
      421 agtaaacccc taccaactgg tcggggtttg aaactatgtg ctggccttgg tgaggtttga
      481 tccgcaagta aacccctacc aactggtcgg ggtttgaaac tttttttgaa ttctgatgcg
      541 gtgaagcttc aagtttgtac aaaaaagcag gcttagttat taatagtaat caattacggg
      601 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      661 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      721 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      781 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      841 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      901 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      961 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     1021 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     1081 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     1141 tggtttagtg aaccgtcaga tcacccagct ttcttgtaca aagtgggcca ccatgagccc
     1201 caagaagaag agaaaggtgg aggccagcat cgaaaaaaaa aagtccttcg ccaagggcat
     1261 gggcgtgaag tccacactcg tgtccggctc caaagtgtac atgacaacct tcgccgaagg
     1321 cagcgacgcc aggctggaaa agatcgtgga gggcgacagc atcaggagcg tgaatgaggg
     1381 cgaggccttc agcgctgaaa tggccgataa aaacgccggc tataagatcg gcaacgccaa
     1441 attcagccat cctaagggct acgccgtggt ggctaacaac cctctgtata caggacccgt
     1501 ccagcaggat atgctcggcc tgaaggaaac tctggaaaag aggtacttcg gcgagagcgc
     1561 tgatggcaat gacaatattt gtatccaggt gatccataac atcctggaca ttgaaaaaat
     1621 cctcgccgaa tacattacca acgccgccta cgccgtcaac aatatctccg gcctggataa
     1681 ggacattatt ggattcggca agttctccac agtgtatacc tacgacgaat tcaaagaccc
     1741 cgagcaccat agggccgctt tcaacaataa cgataagctc atcaacgcca tcaaggccca
     1801 gtatgacgag ttcgacaact tcctcgataa ccccagactc ggctatttcg gccaggcctt
     1861 tttcagcaag gagggcagaa attacatcat caattacggc aacgaatgct atgacattct
     1921 ggccctcctg agcggactga ggcactgggt ggtccataac aacgaagaag agtccaggat
     1981 ctccaggacc tggctctaca acctcgataa gaacctcgac aacgaataca tctccaccct
     2041 caactacctc tacgacagga tcaccaatga gctgaccaac tccttctcca agaactccgc
     2101 cgccaacgtg aactatattg ccgaaactct gggaatcaac cctgccgaat tcgccgaaca
     2161 atatttcaga ttcagcatta tgaaagagca gaaaaacctc ggattcaata tcaccaagct
     2221 cagggaagtg atgctggaca ggaaggatat gtccgagatc aggaaaaatc ataaggtgtt
     2281 cgactccatc aggaccaagg tctacaccat gatggacttt gtgatttata ggtattacat
     2341 cgaagaggat gccaaggtgg ctgccgccaa taagtccctc cccgataatg agaagtccct
     2401 gagcgagaag gatatctttg tgattaacct gaggggctcc ttcaacgacg accagaagga
     2461 tgccctctac tacgatgaag ctaatagaat ttggagaaag ctcgaaaata tcatgcacaa
     2521 catcaaggaa tttaggggaa acaagacaag agagtataag aagaaggacg cccctagact
     2581 gcccagaatc ctgcccgctg gccgtgatgt ttccgccttc agcaaactca tgtatgccct
     2641 gaccatgttc ctggatggca aggagatcaa cgacctcctg accaccctga ttaataaatt
     2701 cgataacatc cagagcttcc tgaaggtgat gcctctcatc ggagtcaacg ctaagttcgt
     2761 ggaggaatac gcctttttca aagactccgc caagatcgcc gatgagctga ggctgatcaa
     2821 gtccttcgct agaatgggag aacctattgc cgatgccagg agggccatgt atatcgacgc
     2881 catccgtatt ttaggaacca acctgtccta tgatgagctc aaggccctcg ccgacacctt
     2941 ttccctggac gagaacggaa acaagctcaa gaaaggcaag cacggcatga gaaatttcat
     3001 tattaataac gtgatcagca ataaaaggtt ccactacctg atcagatacg gtgatcctgc
     3061 ccacctccat gagatcgcca aaaacgaggc cgtggtgaag ttcgtgctcg gcaggatcgc
     3121 tgacatccag aaaaaacagg gccagaacgg caagaaccag atcgacaggt actacgaaac
     3181 ttgtatcgga aaggataagg gcaagagcgt gagcgaaaag gtggacgctc tcacaaagat
     3241 catcaccgga atgaactacg accaattcga caagaaaagg agcgtcattg aggacaccgg
     3301 cagggaaaac gccgagaggg agaagtttaa aaagatcatc agcctgtacc tcaccgtgat
     3361 ctaccacatc ctcaagaata ttgtcaatat caacgccagg tacgtcatcg gattccattg
     3421 cgtcgagcgt gatgctcaac tgtacaagga gaaaggctac gacatcaatc tcaagaaact
     3481 ggaagagaag ggattcagct ccgtcaccaa gctctgcgct ggcattgatg aaactgcccc
     3541 cgataagaga aaggacgtgg aaaaggagat ggctgaaaga gccaaggaga gcattgacag
     3601 cctcgagagc gccaacccca agctgtatgc caattacatc aaatacagcg acgagaagaa
     3661 agccgaggag ttcaccaggc agattaacag ggagaaggcc aaaaccgccc tgaacgccta
     3721 cctgaggaac accaagtgga atgtgatcat cagggaggac ctcctgagaa ttgacaacaa
     3781 gacatgtacc ctgttcagaa acaaggccgt ccacctggaa gtggccaggt atgtccacgc
     3841 ctatatcaac gacattgccg aggtcaattc ctacttccaa ctgtaccatt acatcatgca
     3901 gagaattatc atgaatgaga ggtacgagaa aagcagcgga aaggtgtccg agtacttcga
     3961 cgctgtgaat gacgagaaga agtacaacga taggctcctg aaactgctgt gtgtgccttt
     4021 cggctactgt atccccaggt ttaagaacct gagcatcgag gccctgttcg ataggaacga
     4081 ggccgccaag ttcgacaagg agaaaaagaa ggtgtccggc aattccggat ccggacctaa
     4141 gaaaaagagg aaggtggcgg ccgcttaccc atacgatgtt ccagattacg cttaacaact
     4201 ttattataca tagttggaat tcctagagct cgctgatcag cctcgactgt gccttctagt
     4261 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     4321 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     4381 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc
     4441 aggcatgctg gggagggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     4501 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4561 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     4621 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     4681 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4741 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4801 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4861 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4921 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4981 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     5041 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5101 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     5161 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     5221 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5281 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     5341 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5401 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5461 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5521 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5581 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5641 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5701 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5761 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5821 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5881 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5941 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6001 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6061 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6121 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6181 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg
     6241 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6301 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6361 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6421 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6481 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6541 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6601 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6661 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6721 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6781 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6841 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6901 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6961 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7021 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7081 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7141 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
