LOCUS       sequence-463539-        5767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 guide RNA targeting GAA repeats for A-to-G base editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5767)
  AUTHORS   Matuszek Z, Arbab M, Kesavan M, Hsu A, Roy JCL, Zhao J, Yu T, 
            Weisburd B, Newby GA, Doherty NJ, Wu M, Shibata S, Cristian A, Tao 
            YA, Fearnley LG, Bahlo M, Rehm HL, Xie J, Gao G, Mouro Pinto R, Liu 
            DR
  TITLE     Base editing of trinucleotide repeats that cause Huntington's 
            disease and Friedreich's ataxia reduces somatic repeat expansions in
            patient cells and in mice.
  JOURNAL   Nat Genet. 2025 May 26. doi: 10.1038/s41588-025-02172-8.
  PUBMED    40419681
REFERENCE   2  (bases 1 to 5767)
  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: "Nat Genet. 
            2025 May 26. doi: 10.1038/s41588-025-02172-8."
COMMENT     Sequence Label: p2T-U6-sgGAA
FEATURES             Location/Qualifiers
     source          1..5767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     241..260
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     494..511
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    628..649
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(664..669,670..687,688..694)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    702..718
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     707..729
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     726..742
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     726..742
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     761..781
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        763..781
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    join(838..849,850..1037)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(1047..1067)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1091..1301
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        1113..1309
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      1160..1295
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1222..1241
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1358..2383
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1358..2383
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    2513..2634
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2758..2879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(3102..3350)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3160..3179)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3330..3350)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     protein_bind    3368..3401
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3439..3472
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3478..3487
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(3512..3674,3675..3686)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3716..3735)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3717..3735)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3745..3762)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3745..3761)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3754..3776)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3903..4357
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3989..4008)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4199..4220
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4383..4487
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4488..4556,4557..5348)
                     /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(4706..4725)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5519..5767,1..340)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
       61 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      121 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      181 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
      241 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
      301 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
      361 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
      421 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
      481 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
      541 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
      601 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      661 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
      721 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
      781 ggaacaaaag ctggagctcg gatctgctgg gcttgctgaa ggtagggggt caagaaccag
      841 aggtgtaaag tacttgagta attttacttg attactgtac ttaagtatta tttttgggga
      901 tttttacttt acttgagtac aattaaaaat caatactttt acttttactt aattacattt
      961 ttttagaaaa aaaagtactt tttactcctt acaattttat ttacagtcaa aaagtactta
     1021 ttttttggag atcacttgaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     1081 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     1141 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     1201 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     1261 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     1321 agcttgtata tccattttcg gatctgatca gcacgtgatg aaaaagcctg aactcaccgc
     1381 gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct
     1441 ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct
     1501 gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc
     1561 atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac
     1621 ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact
     1681 gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag
     1741 ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg
     1801 tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga
     1861 caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg
     1921 ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa
     1981 tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga
     2041 ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta
     2101 cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg
     2161 cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg
     2221 ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca
     2281 aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag
     2341 tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagcacgtgc tacgagattt
     2401 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     2461 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     2521 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2581 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     2641 ctgtataccg tcgaccttcg cgattttacc acatttgtag aggttttact tgctttaaaa
     2701 aacctcccac atctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     2761 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2821 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     2881 catgtctgct cgaagcggcc gtacctctag agccatttgt ctgcagaatt ggcgcacgcg
     2941 ctaaaaacgg actagcctta ttttaacttg ctatttctag ctctaaaaca aaaaagcacc
     3001 gactcggtgc cactttttca agttgataac ggactagcct tatttaaact tgctatgctg
     3061 tttccagcat agctcttaaa ctcttcttct tcttcttctt cggtgtttcg tcctttccac
     3121 aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc
     3181 attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac
     3241 gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat ctctctaaca
     3301 gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc acatgcgcgc
     3361 cggtaccata acttcgtata aagtatccta tacgaagtta tggccggcca agggcaattc
     3421 caccacactg gactatcgat aacttcgtat aatgtatgct atacgaagtt atcgacacca
     3481 tggtggcctc cagatccttc gagatcgatc tttcttgctt ttacttttac ttccttaata
     3541 ctcaagtaca attttaatgg agtacttttt tacttttact caagtaagat tctagccaga
     3601 tacttttact tttaattgag taaaattttc cctaagtact tgtactttca cttgagtaaa
     3661 atttttgagt actttttaca cctctgtcaa gaaccatatg ccggtaccca attcgcccta
     3721 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     3781 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     3841 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     3901 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3961 caccacaccc gccgcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4021 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4081 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4141 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4201 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4261 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4321 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     4381 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4441 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4501 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4561 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4621 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4681 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4741 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4801 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4861 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4921 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4981 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5041 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5101 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5161 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5221 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5281 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5341 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5401 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5461 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5521 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5581 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5641 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5701 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5761 ccagtgg
//
