LOCUS       sequence-345452-        2736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing paired pegRNAs from human U6 and H1 promoters to make 
            10204-bp deletion on HPRT1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2736)
  AUTHORS   Choi J, Chen W, Suiter CC, Lee C, Chardon FM, Yang W, Leith A, Daza 
            RM, Martin B, Shendure J
  TITLE     Precise genomic deletions using paired prime editing.
  JOURNAL   Nat Biotechnol. 2021 Oct 14. pii: 10.1038/s41587-021-01025-z. doi: 
            10.1038/s41587-021-01025-z.
  PUBMED    34650269
REFERENCE   2  (bases 1 to 2736)
  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 
            Biotechnol. 2021 Oct 14. pii: 10.1038/s41587-021-01025-z. doi: 
            10.1038/s41587-021-01025-z."
COMMENT     Sequence Label: pU6nH1-HD10kb
FEATURES             Location/Qualifiers
     source          1..2736
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(153..170)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(153..169)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(162..184)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_RNA        complement(237..312)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(334..582)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(392..411)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(562..582)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        689..793
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(794..862,863..1654)
                     /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(1012..1031)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1825..2413
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2314..2333
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(2562..2637)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(join(2728..2736,1..11))
                     /label=H1
                     /note="Human H1 promoter, forward primer"
ORIGIN
        1 acacatagcg acatgcaaat attgcagggc gccactcccc tgtccctcac agccatcttc
       61 ctgccagggc gcacgcgcgc tgggtgttcc cgcctagtga cactgggccc gcgattcctt
      121 ggagcgggtt gatgacgtca gcgttcgaat tcactggccg tcgttttaca acgtcgtgac
      181 tgggaaaaaa atggcccgta ataccctacc agtttgcagg ctcactaggt agccgtgcac
      241 cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac ttgctatttc
      301 tagctctaaa accaggtatt acgggccaac ctcggtgttt cgtcctttcc acaagatata
      361 taaagccaag aaatcgaaat actttcaagt tacggtaagc atatgatagt ccattttaaa
      421 acataatttt aaaactgcaa actacccaag aaattattac tttctacgtc acgtattttg
      481 tactaatatc tttgtgttta cagtcaaatt aattctaatt atctctctaa cagccttgta
      541 tcgtatatgc aaatatgaag gaatcatggg aaataggccc tcttcctgcc cgaccttggt
      601 accggatcca gtcgactgaa ttggttcctt taaagcctgc ttttttgtac agctagcgac
      661 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
      721 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
      781 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
      841 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
      901 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
      961 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     1021 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     1081 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     1141 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     1201 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     1261 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     1321 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     1381 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     1441 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     1501 tgagcgtgga tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     1561 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     1621 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     1681 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1741 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1801 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1861 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1921 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     1981 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2041 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2101 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2161 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2221 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2281 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2341 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2401 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2461 ttttgctcac atgtcatatg tagagaggta cctcgagcgg cccaagctta aaaaaagcaa
     2521 actggtaggg tattacgggc caacctgaca atacatgaag tgcaccgact cggtgccact
     2581 ttttcaagtt gataacggac tagccttatt ttaacttgct atttctagct ctaaaacgcg
     2641 cctaccagtt tgcaggctgg ggatctgtgg tcctcataca gaacttataa gattcccaaa
     2701 tccaaagaca tttcacgttt atggtgattt cccaga
//
