LOCUS       sequence-462760-        3478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pegRNA with optimized 3' modifications to correct the rd6 mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3478)
  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 3478)
  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_rd6_pegRNA_(PP7-C4-Q1)
FEATURES             Location/Qualifiers
     source          1..3478
                     /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    477..496
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(574..593)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(575..593)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(603..620)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(603..619)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(612..634)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      761..1216
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(848..867)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1058..1079
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1242..1346
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1347..1415,1416..2207)
                     /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(1565..1584)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2378..2966
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2867..2886
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3120..3137
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3254..3275
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3290..3295,3296..3313,3314..3320)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3328..3344
                     /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     3333..3355
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3352..3368
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3352..3368
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3387..3407
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3389..3407
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    3431..3478
                     /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 aacaccgctt
      301 ctccagagag tgcccttgtt tcagagctat gctggaaaca gcatagcaag ttgaaataag
      361 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctaccaga ccagtaagtc
      421 ccaagggcac tctctggcaa ttccgaccag aagatatggc ttcggttgga attgaagttc
      481 actgccgtat aggcagctaa gaaacgcggt tctatctagt tacgcgttaa accaactaga
      541 attttttact agttctagag cggccccaat tcgccctata gtgagtcgta ttacgcgcgc
      601 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
      661 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
      721 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
      781 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgacca ccacacttgc cagcgcccta
      841 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
      901 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
      961 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     1021 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     1081 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     1141 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     1201 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1261 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1321 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1381 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1441 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     1501 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1561 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     1621 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     1681 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     1741 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     1801 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     1861 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     1921 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     1981 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2041 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     2101 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2161 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2221 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2281 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2341 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2401 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2461 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2521 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2581 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2641 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2701 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2761 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2821 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2881 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2941 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3001 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3061 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3121 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     3181 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     3241 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     3301 tgctcccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     3361 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
     3421 aaacatgcat gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttc
//
