LOCUS       sequence-451253-        2280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pUC19 plasmid with U6 promoter and SpCas9 gRNA to create an 
            OPA1-del_exon5 cell line via nuclease mediate excision, gRNA 2b must
            be used with gRNA 1a or 1b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2280)
  AUTHORS   Fry MY, Navarro PP, Hakim P, Ananda VY, Qin X, Landoni JC, Rath S, 
            Inde Z, Lugo CM, Luce BE, Ge Y, McDonald JL, Ali I, Ha LL, 
            Kleinstiver BP, Chan DC, Sarosiek KA, Chao LH
  TITLE     In situ architecture of Opa1-dependent mitochondrial cristae 
            remodeling.
  JOURNAL   EMBO J. 2024 Feb;43(3):391-413. doi: 10.1038/s44318-024-00027-2. 
            Epub 2024 Jan 15.
  PUBMED    38225406
REFERENCE   2  (bases 1 to 2280)
  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: 
            "10.1038/s44318-024-00027-2"; journalName: "EMBO J"; date: 
            "2024-02"; volume: "43"; issue: "3"; pages: "391-413"
COMMENT     Sequence Label: pUC19-U6-SpCas9_gRNA-OPA1-del_exon5-gRNA2b_(CJ...
FEATURES             Location/Qualifiers
     source          1..2280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     82..102
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     253..272
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        352..427
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(531..1119)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(611..630)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1290..2081,2082..2150))
                     /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     1913..1932
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2151..2255)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc
       61 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      121 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      181 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      241 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      301 ttatatatct tgtggaaagg acgaaacacc gcagaagcgc aaggtgatgg agttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      421 tcggtgcttt ttttaagctt gggccgctcg aggtacctct ctacatatga catgtgagca
      481 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
      541 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
      601 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
      661 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
      721 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
      781 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
      841 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
      901 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
      961 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     1021 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     1081 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     1141 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     1201 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     1261 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     1321 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     1381 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     1441 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     1501 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     1561 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     1621 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     1681 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     1741 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     1801 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     1861 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     1921 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     1981 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     2041 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     2101 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     2161 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     2221 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
//
