LOCUS       sequence-380385-        5162 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express CasRX gRNA and contains MCS to insert coding region of 
            interest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5162)
  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 5162)
  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)-CMV-intron-MCS-pA
FEATURES             Location/Qualifiers
     source          1..5162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      9..597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     498..517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   659..788
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        803..1051
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     803..823
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     974..993
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1053..1082
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     enhancer        1182..1485
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1486..1689
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1640..1660
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1686..1710
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1838..2216
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     polyA_signal    2311..2787
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2510..2529)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     polyA_signal    2835..2969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2859..2878)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2913..2932
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3083..3223
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3083..3212
                     /label=AAV2 ITR
     rep_origin      3298..3753
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3385..3404)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3595..3616
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3770..3789)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3889..3911
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3949..3967)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4035..4139
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4140..4208,4209..5000)
                     /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(4358..4377)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
       61 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
      121 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
      181 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
      241 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
      301 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
      361 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
      421 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
      481 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
      541 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
      601 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc
      661 tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg
      721 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
      781 gggttcctgc ggccgcacta gcgagggcct atttcccatg attccttcat atttgcatat
      841 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      901 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      961 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     1021 tttatatatc ttgtggaaag gacgaaacac cgaaccccta ccaactggtc ggggtttgaa
     1081 acggaagagc gagctcttct tttttttctg tacaacgcgt ctagttatta atagtaatca
     1141 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     1201 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1261 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1321 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     1381 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     1441 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     1501 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     1561 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     1621 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     1681 agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
     1741 ctccatagaa gacaccggga ccgatccagc ctccgcggat tcgaatcccg gccgggaacg
     1801 gtgcattgga acgcggattc cccgtgccaa gagtgacgta agtaccgcct atagagtcta
     1861 taggcccaca aaaaatgctt tcttctttta atatactttt ttgtttatct tatttctaat
     1921 actttcccta atctctttct ttcagggcaa taatgataca atgtatcatg cctctttgca
     1981 ccattctaaa gaataacagt gataatttct gggttaaggc aatagcaata tttctgcata
     2041 taaatatttc tgcatataaa ttgtaactga tgtaagaggt ttcatattgc taatagcagc
     2101 tacaatccag ctaccattct gcttttattt tatggttggg ataaggctgg attattctga
     2161 gtccaagcta ggcccttttg ctaatcatgt tcatacctct tatcttcctc ccacagctcc
     2221 tgggcaacgt gctggtctgt gtgctggccc atcactttgg caaagaattg ggattcgaac
     2281 atcgattgaa ttctgcagtc ggggatctac gggtggcatc cctgtgaccc ctccccagtg
     2341 cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta
     2401 agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg
     2461 gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa
     2521 ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa
     2581 gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc
     2641 agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc
     2701 aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg
     2761 tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccgggcc caatggcgcg
     2821 ctagataata cgtagatcca gacatgataa gatacattga tgagtttgga caaaccacaa
     2881 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     2941 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     3001 aggttcaggg ggaggtgtgg gaggtttttt aattcttgcg gccgcggcgg taccggtaac
     3061 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     3121 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     3181 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     3241 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     3301 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3361 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3421 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3481 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     3541 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3601 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     3661 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3721 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     3781 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     3841 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     3901 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     3961 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4021 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4081 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4141 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4201 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4261 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4321 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4381 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4441 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4501 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4561 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4621 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4681 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4741 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4801 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4861 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4921 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4981 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5041 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5101 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5161 aa
//
