LOCUS       sequence-462756-        3470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pegRNA with optimized 3' modifications to facilitate a +1 T to A 
            prime edit in the HEK3 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3470)
  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 3470)
  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_HEK3+1T>A_(PP7-C4-Q1)
FEATURES             Location/Qualifiers
     source          1..3470
                     /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    469..488
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(566..585)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(567..585)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(595..612)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(595..611)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(604..626)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      753..1208
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(840..859)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1050..1071
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1234..1338
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1339..1407,1408..2199)
                     /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(1557..1576)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2370..2958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2859..2878
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3112..3129
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3246..3267
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3282..3287,3288..3305,3306..3312)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3320..3336
                     /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     3325..3347
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3344..3360
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3344..3360
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3379..3399
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3381..3399
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    3423..3470
                     /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 aacaccggcc
      301 cagactgagc acgtgagttt cagagctatg ctggaaacag catagcaagt tgaaataagg
      361 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttcctctg ccatctcgtg
      421 ctcagtctgc aattccgacc agaagatatg gcttcggttg gaattgaagt tcactgccgt
      481 ataggcagct aagaaacgcg gttctatcta gttacgcgtt aaaccaacta gaatttttta
      541 ctagttctag agcggcccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
      601 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
      661 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
      721 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
      781 ggcgggtgtg gtggttacgc gcagcgtgac caccacactt gccagcgccc tagcgcccgc
      841 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
      901 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
      961 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1021 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1081 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     1141 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     1201 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1261 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     1321 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1381 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1441 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1501 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     1561 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     1621 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     1681 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     1741 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     1801 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     1861 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     1921 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     1981 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2041 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2101 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     2161 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     2221 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     2281 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2341 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2401 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2461 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     2521 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2581 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2641 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2701 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2761 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2821 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2881 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2941 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3001 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3061 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3121 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     3181 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     3241 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgctcccg
     3301 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     3361 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggaaacatgc
     3421 atgaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc
//
