LOCUS       sequence-486263-        2380 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Mammalian expression of epegRNA for twin prime editing of half the 
            minimal PhiC31 attP site targeting the human ROSA26 site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2380)
  AUTHORS   Hew BE, Gupta S, Sato R, Waller DF, Stoytchev I, Short JE, Sharek L,
            Tran CT, Badran AH, Owens JB
  TITLE     Directed evolution of hyperactive integrases for site specific 
            insertion of transgenes.
  JOURNAL   Nucleic Acids Res. 2024 Jul 2:gkae534. doi: 10.1093/nar/gkae534.
  PUBMED    38953167
REFERENCE   2  (bases 1 to 2380)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2024 Jul 2:gkae534. doi: 10.1093/nar/gkae534."
COMMENT     Sequence Label: pU6 pegRNA ROSA 4a PhiC31 attP41
FEATURES             Location/Qualifiers
     source          1..2380
                     /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"
     rep_origin      complement(631..1219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(711..730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1390..2181,2182..2250))
                     /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     2013..2032
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2251..2355)
                     /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 atgcctggta gggatgcaaa gtttaagagc
      361 taagctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac tcgaaagagt
      421 ggcaccgagt cggtgctggg gtaacctttg agttctctca gttgggggcg catccctacc
      481 agatctaatg cgcggttcta tctagttacg cgttaaacca actagaattt ttttaagctt
      541 gggccgctcg aggtacctct ctacatatga catgtgagca aaaggccagc aaaaggccag
      601 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
      661 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
      721 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
      781 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
      841 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
      901 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
      961 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     1021 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     1081 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     1141 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     1201 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     1261 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     1321 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1381 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1441 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     1501 atctggcccc agtgctgcaa tgataccgcg agatccacgc tcaccggctc cagatttatc
     1561 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1621 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1681 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1741 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     1801 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     1861 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     1921 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     1981 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     2041 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     2101 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     2161 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     2221 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     2281 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     2341 aataggggtt ccgcgcacat ttccccgaaa agtgccacct
//
