LOCUS       sequence-462774-        3346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  nsgRNA for PE3b correction of the rd6 mutation, contains a PP7 in 
            the scaffold tetraloop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3346)
  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 3346)
  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_nsgRNA(PP7)
FEATURES             Location/Qualifiers
     source          1..3346
                     /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"
     primer_bind     complement(442..461)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(443..461)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(471..488)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(471..487)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(480..502)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      629..1084
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(716..735)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     926..947
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1110..1214
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1215..1283,1284..2075)
                     /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(1433..1452)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2246..2834
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2735..2754
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2988..3005
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3122..3143
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3158..3163,3164..3181,3182..3188)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3196..3212
                     /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     3201..3223
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3220..3236
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3220..3236
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3255..3275
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3257..3275
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    3299..3346
                     /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 aacaccgcac
      301 cagaccagta agtcccagtt tcagagccac cagaagatat ggcttcggtg gcaagttgaa
      361 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttactag
      421 ttctagagcg gccccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
      481 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
      541 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
      601 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
      661 ggtgtggtgg ttacgcgcag cgtgaccacc acacttgcca gcgccctagc gcccgctcct
      721 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
      781 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
      841 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
      901 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
      961 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1021 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     1081 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1141 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1201 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1261 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1321 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1381 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1441 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1501 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1561 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1621 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1681 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1741 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1801 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1861 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1921 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1981 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2041 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2101 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2161 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2221 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2281 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2341 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2401 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2461 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2521 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2581 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2641 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2701 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2761 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2821 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2881 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2941 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3001 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     3061 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     3121 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg ctcccggctc
     3181 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     3241 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggaa acatgcatga
     3301 agttcctatt ccgaagttcc tattctctag aaagtatagg aacttc
//
