LOCUS       sequence-334032-        3293 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express a nicking sgRNA used for correction (via A•T-to-G•C) of the 
            E342K mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3293)
  AUTHORS   Liu P, Liang SQ, Zheng C, Mintzer E, Zhao YG, Ponnienselvan K, Mir 
            A, Sontheimer EJ, Gao G, Flotte TR, Wolfe SA, Xue W
  TITLE     Improved prime editors enable pathogenic allele correction and 
            cancer modelling in adult mice.
  JOURNAL   Nat Commun. 2021 Apr 9;12(1):2121. doi: 10.1038/s41467-021-22295-w.
  PUBMED    33837189
REFERENCE   2  (bases 1 to 3293)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-04-9"; volume: "12(1):2121. doi"; pages: " 
            10.1038/s41467-021-22295-w"
COMMENT     Sequence Label: sgAAT Nicking
FEATURES             Location/Qualifiers
     source          1..3293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        13..117
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(118..186,187..978)
                     /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(336..355)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1149..1737
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1638..1657
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1891..1908
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2025..2046
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2061..2066,2067..2084,2085..2091)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2099..2115
                     /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     2104..2126
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2123..2139
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2123..2139
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2158..2178
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2160..2178
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2225..2473
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2225..2245
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2396..2415
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2494..2569
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2597..2613)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2598..2614)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2638..2657)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2639..2657)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2667..2684)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2667..2683)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2676..2698)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2825..3280
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2912..2931)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3122..3143
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
       61 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
      121 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
      181 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
      241 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
      301 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      361 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      421 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      481 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
      541 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
      601 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
      661 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
      721 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
      781 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
      841 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
      901 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
      961 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     1021 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     1081 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1141 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1201 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1261 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     1321 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1381 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1441 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1501 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     1561 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     1621 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     1681 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     1741 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     1801 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     1861 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     1921 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     1981 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     2041 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     2101 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
     2161 attaaccctc actaaaggga acaaaagctg gagctccacc gcggtggcgg ccgccccctt
     2221 caccgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     2281 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     2341 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     2401 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     2461 aggacgaaac accgggtttg ttgaacttga cctgttttag agctagaaat agcaagttaa
     2521 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttgaat
     2581 tcgatatcaa gcttatcgat accgtcgacc tcgagggggg gcccggtacc caattcgccc
     2641 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     2701 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2761 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     2821 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2881 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2941 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3001 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3061 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     3121 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     3181 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     3241 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact ttt
//
