LOCUS       sequence-462757-        3345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  nsgRNA for PE3 facilitation of a +1 T to A prime edit on the HEK3 
            locus, contains a PP7 in the scaffold tetraloop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3345)
  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 3345)
  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_nsgRNA(PP7)
FEATURES             Location/Qualifiers
     source          1..3345
                     /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(441..460)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(442..460)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(470..487)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(470..486)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(479..501)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      628..1083
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(715..734)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     925..946
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1109..1213
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1214..1282,1283..2074)
                     /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(1432..1451)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2245..2833
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2734..2753
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2987..3004
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3121..3142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3157..3162,3163..3180,3181..3187)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3195..3211
                     /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     3200..3222
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3219..3235
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3219..3235
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3254..3274
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3256..3274
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    3298..3345
                     /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 aacaccgtca
      301 accagtatcc cggtgcgttt cagagccacc agaagatatg gcttcggtgg caagttgaaa
      361 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttactagt
      421 tctagagcgg ccccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
      481 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
      541 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
      601 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
      661 gtgtggtggt tacgcgcagc gtgaccacca cacttgccag cgccctagcg cccgctcctt
      721 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
      781 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
      841 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
      901 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
      961 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1021 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     1081 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1141 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1201 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1261 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     1321 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     1381 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     1441 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     1501 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     1561 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     1621 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     1681 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     1741 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     1801 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     1861 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     1921 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     1981 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2041 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2101 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2161 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2221 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2281 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2341 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     2401 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2461 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2521 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2581 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2641 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2701 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2761 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2821 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2881 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2941 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3001 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     3061 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     3121 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc tcccggctcg
     3181 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     3241 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggaaa catgcatgaa
     3301 gttcctattc cgaagttcct attctctaga aagtatagga acttc
//
