LOCUS       sequence-486390-        2381 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 2381)
  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 2381)
  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 4b PhiC31 attP41
FEATURES             Location/Qualifiers
     source          1..2381
                     /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(632..1220)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(712..731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1391..2182,2183..2251))
                     /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     2014..2033
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2252..2356)
                     /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 caccaccaaa gtgtagcatc gtttaagagc
      361 taagctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac tcgaaagagt
      421 ggcaccgagt cggtgcactg agagaactca aaggttaccc cagttggggc gctacacttt
      481 ggttaataac gcgcggttct atctagttac gcgttaaacc aactagaatt tttttaagct
      541 tgggccgctc gaggtacctc tctacatatg acatgtgagc aaaaggccag caaaaggcca
      601 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
      661 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
      721 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
      781 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
      841 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
      901 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
      961 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     1021 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     1081 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     1141 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     1201 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     1261 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     1321 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     1381 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     1441 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     1501 catctggccc cagtgctgca atgataccgc gagatccacg ctcaccggct ccagatttat
     1561 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     1621 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     1681 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     1741 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     1801 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     1861 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     1921 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     1981 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     2041 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     2101 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     2161 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     2221 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     2281 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     2341 aaataggggt tccgcgcaca tttccccgaa aagtgccacc t
//
