LOCUS       sequence-486267-        2376 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of epegRNA for twin prime editing of half the 
            minimal Bxb1 attB site targeting the human ROSA26 site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2376)
  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 2376)
  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: "Nucleic 
            Acids Res. 2024 Jul 2:gkae534. doi: 10.1093/nar/gkae534."
COMMENT     Sequence Label: pU6 pegRNA ROSA 4a Bxb1 attB38
FEATURES             Location/Qualifiers
     source          1..2376
                     /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(627..1215)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(707..726)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1386..2177,2178..2246))
                     /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     2009..2028
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2247..2351)
                     /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 cggtgcacga cggcggtctc cgtcgtcagg atcatgcatc cctaccagaa
      481 cttcgtcgcg gttctatcta gttacgcgtt aaaccaacta gaattttttt aagcttgggc
      541 cgctcgaggt acctctctac atatgacatg tgagcaaaag gccagcaaaa ggccaggaac
      601 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
      661 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
      721 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
      781 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
      841 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
      901 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
      961 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1021 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     1081 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     1141 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     1201 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     1261 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     1321 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     1381 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     1441 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     1501 ggccccagtg ctgcaatgat accgcgagat ccacgctcac cggctccaga tttatcagca
     1561 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     1621 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     1681 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     1741 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     1801 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     1861 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     1921 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     1981 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     2041 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     2101 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     2161 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     2221 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     2281 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     2341 ggggttccgc gcacatttcc ccgaaaagtg ccacct
//
