LOCUS       sequence-450172-        2306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA and template for MET 5'UTR uORF2 mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2306)
  AUTHORS   Cerqua M, Foiani M, Boccaccio C, Comoglio PM, Altintas DM
  TITLE     The integrated stress response drives MET oncogene overexpression in
            cancers.
  JOURNAL   EMBO J. 2025 Jan 7. doi: 10.1038/s44318-024-00338-4.
  PUBMED    39774381
REFERENCE   2  (bases 1 to 2306)
  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"; journalName: "EMBO J. 
            2025 Jan 7. doi: 10.1038/s44318-024-00338-4."
COMMENT     Sequence Label: pU6-sgRNA_MET_uORF2
FEATURES             Location/Qualifiers
     source          1..2306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..249)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(59..78)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(229..249)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        356..460
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(461..529,530..1321)
                     /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(679..698)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1492..2080
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1981..2000
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(2211..2286)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
       61 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
      121 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
      181 tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
      241 taggccctct tcctgcccga ccttggtacc ggatccagtc gactgaattg gttcctttaa
      301 agcctgcttt tttgtacagc tagcgacgtc aggtggcact tttcggggaa atgtgcgcgg
      361 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
      421 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
      481 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
      541 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
      601 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
      661 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
      721 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
      781 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
      841 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
      901 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
      961 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     1021 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     1081 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     1141 gtttattgct gataaatctg gagccggtga gcgtggatct cgcggtatca ttgcagcact
     1201 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     1261 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     1321 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     1381 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     1441 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     1501 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     1561 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     1621 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     1681 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     1741 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     1801 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     1861 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1921 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1981 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2041 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2101 tttacggttc ctggcctttt gctggccttt tgctcacatg tcatatgtag agaggtacct
     2161 cgagcggccc aagcttaaaa aaagctttgt gagcagttcc ggagccgagt gcaccgactc
     2221 ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta tttctagctc
     2281 taaaaccatc tgctcacaaa gcgctc
//
