LOCUS       sequence-462754-        3343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty gRNA backbone which contains the PP7 aptamer in the scaffold 
            tetraloop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3343)
  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 3343)
  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_empty-sgRNA(PP7)
FEATURES             Location/Qualifiers
     source          1..3343
                     /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(439..458)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(440..458)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(468..485)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(468..484)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(477..499)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      626..1081
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(713..732)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     923..944
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1107..1211
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1212..1280,1281..2072)
                     /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(1430..1449)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2243..2831
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2732..2751
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2985..3002
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3119..3140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3155..3160,3161..3178,3179..3185)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3193..3209
                     /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     3198..3220
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3217..3233
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3217..3233
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3252..3272
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3254..3272
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    3296..3343
                     /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 aacaccgggt
      301 cttcgagaag acctgtttca gagccaccag aagatatggc ttcggtggca agttgaaata
      361 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttactagttc
      421 tagagcggcc ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
      481 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      541 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      601 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
      661 gtggtggtta cgcgcagcgt gaccaccaca cttgccagcg ccctagcgcc cgctcctttc
      721 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      781 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      841 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
      901 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
      961 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     1021 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     1081 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1141 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1201 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1261 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1321 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1381 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     1441 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1501 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1561 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1621 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1681 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1741 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1801 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1861 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     1921 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     1981 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2041 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2101 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2161 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2221 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2281 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2341 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     2401 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2461 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2521 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2581 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2641 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2701 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2761 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2821 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2881 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2941 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3001 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     3061 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     3121 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctc ccggctcgta
     3181 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     3241 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggaaaca tgcatgaagt
     3301 tcctattccg aagttcctat tctctagaaa gtataggaac ttc
//
