LOCUS       sequence-462758-        3463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pegRNA with optimized 3' modifications to correct the rd12 mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3463)
  AUTHORS   Geilenkeuser J, Armbrust N, Steinmassl E, Du SW, Schmidt S, Binder 
            EMH, Li Y, Warsing NW, Wendel SV, von der Linde F, Schiele EM, Niu 
            X, Stroppel L, Berezin O, Santl TH, Orschmann T, Nelson K, Gruber C,
            Palczewska G, Menezes CR, Risaliti E, Engfer ZJ, Koleci N, Schmidts 
            A, Geerlof A, Palczewski K, Westmeyer GG, Truong DJ
  TITLE     Engineered nucleocytosolic vehicles for loading of programmable 
            editors.
  JOURNAL   Cell. 2025 May 15;188(10):2637-2655.e31. doi: 
            10.1016/j.cell.2025.03.015. Epub 2025 Apr 9.
  PUBMED    40209705
REFERENCE   2  (bases 1 to 3463)
  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"; doi: 
            "10.1016/j.cell.2025.03.015"; journalName: "Cell"; date: 
            "2025-05-15- 15"; volume: "188"; issue: "10"; pages: "2637-2655.e31"
COMMENT     Sequence Label: pU6_rd12_pegRNA_(PP7-C4-Q1)
FEATURES             Location/Qualifiers
     source          1..3463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        48..296
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     48..68
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     219..238
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    462..481
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(559..578)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(560..578)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(588..605)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(588..604)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(597..619)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      746..1201
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(833..852)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1043..1064
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1227..1331
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1332..1400,1401..2192)
                     /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(1550..1569)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2363..2951
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2852..2871
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3105..3122
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3239..3260
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3275..3280,3281..3298,3299..3305)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3313..3329
                     /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     3318..3340
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3337..3353
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3337..3353
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3372..3392
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3374..3392
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    3416..3463
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 ataaaacctg caggcatgca agcgatcgcg gggccgcccc cttcaccgag ggcctatttc
       61 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
      121 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
      181 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
      241 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggag
      301 ccctggccca catcaggttt cagagctatg ctggaaacag catagcaagt tgaaataagg
      361 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctagtctcc tccgatgtgg
      421 gccaattccg accagaagat atggcttcgg ttggaattga agttcactgc cgtataggca
      481 gctaagaaac gcggttctat ctagttacgc gttaaaccaa ctagaatttt ttactagttc
      541 tagagcggcc ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
      601 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      661 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      721 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
      781 gtggtggtta cgcgcagcgt gaccaccaca cttgccagcg ccctagcgcc cgctcctttc
      841 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      901 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      961 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     1021 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     1081 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1141 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     1201 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1261 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1321 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1381 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1441 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1501 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     1561 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1621 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1681 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1741 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1801 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1861 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1921 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1981 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2041 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2101 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2161 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2221 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2281 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2341 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2401 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2461 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     2521 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2581 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2641 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2701 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2761 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2821 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2881 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2941 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3001 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3061 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3121 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     3181 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     3241 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctc ccggctcgta
     3301 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     3361 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggaaaca tgcatgaagt
     3421 tcctattccg aagttcctat tctctagaaa gtataggaac ttc
//
