LOCUS       sequence-390154-        3226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  nicking sgRNA to induce or correct GBA (c. 1226 A > G) mutation 
            using PE3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3226)
  AUTHORS   Li M, Zhong A, Wu Y, Sidharta M, Beaury M, Zhao X, Studer L, Zhou T
  TITLE     Transient inhibition of p53 enhances prime editing and cytosine 
            base-editing efficiencies in human pluripotent stem cells.
  JOURNAL   Nat Commun. 2022 Oct 27;13(1):6354. doi: 10.1038/s41467-022-34045-7.
  PUBMED    36302757
REFERENCE   2  (bases 1 to 3226)
  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 
            Commun."; date: "2022-10-27"; volume: "13(1):6354. doi"; pages: " 
            10.1038/s41467-022-34045-7"
COMMENT     Sequence Label: Lsg-GBA-1 + 2
FEATURES             Location/Qualifiers
     source          1..3226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        18..266
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     18..38
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_RNA        288..363
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(397..417)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(397..415)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(436..452)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(436..452)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(449..471)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    460..476
                     /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)."
     promoter        complement(join(484..490,491..508,509..514))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    529..550
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(667..684)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(838..1426)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(918..937)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1597..2388,2389..2457))
                     /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     2220..2239
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2458..2562)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2588..3043)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2725..2746)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2937..2956
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3170..3192
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3184..3201
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3185..3201
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(3211..3226,1..4)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(3211..3226,1..3)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 agggcgaatt gggtaccgag ggcctatttc ccatgattcc ttcatatttg catatacgat
       61 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
      121 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
      181 ttaaaatgga ctatcatatg cttatcgtaa cttgaaagta tttcgatttc ttggctttat
      241 atatcttgtg gaaaggacga aacaccgacc cttacctaca ctctctggtt ttagagctag
      301 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
      361 tgcttttttt ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg
      421 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
      481 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
      541 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
      601 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
      661 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
      721 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
      781 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      841 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
      901 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
      961 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1021 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1081 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1141 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1201 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     1261 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     1321 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1381 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1441 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     1501 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     1561 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     1621 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     1681 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     1741 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     1801 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     1861 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     1921 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     1981 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     2041 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     2101 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     2161 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     2221 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     2281 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     2341 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     2401 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     2461 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2521 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     2581 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     2641 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     2701 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     2761 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     2821 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     2881 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     2941 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     3001 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     3061 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     3121 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     3181 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactat
//
