LOCUS       sequence-437336-        2368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pegRNA 2 plasmid used for PASSIGE-mediated Bxb1 attP installation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2368)
  AUTHORS   Pandey S, Gao XD, Krasnow NA, McElroy A, Tao YA, Duby JE, Steinbeck 
            BJ, McCreary J, Pierce SE, Tolar J, Meissner TB, Chaikof EL, Osborn 
            MJ, Liu DR
  TITLE     Efficient site-specific integration of large genes in mammalian 
            cells via continuously evolved recombinases and prime editing.
  JOURNAL   Nat Biomed Eng. 2024 Jun 10. doi: 10.1038/s41551-024-01227-1.
  PUBMED    38858586
REFERENCE   2  (bases 1 to 2368)
  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: "Nat Biomed 
            Eng. 2024 Jun 10. doi: 10.1038/s41551-024-01227-1."
COMMENT     Sequence Label: AAVS1 pegRNA2 with trimmed attP
FEATURES             Location/Qualifiers
     source          1..2368
                     /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        351..426
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(619..1207)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(699..718)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1378..2169,2170..2238))
                     /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     2001..2020
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2239..2343)
                     /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 gcgactcctg gaagtggcca gttttagagc
      361 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
      421 cggtgcgtca accaccgcgg tctcagtggt gtacggtaca aacctccact tccaggagcg
      481 cggttctatc tagttacgcg ttaaaccaac tagaattttt ttaagcttgg gccgctcgag
      541 gtacctctct acatatgaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
      601 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
      661 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      721 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      781 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
      841 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
      901 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      961 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     1021 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     1081 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1141 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     1201 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     1261 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     1321 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     1381 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     1441 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     1501 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     1561 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     1621 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     1681 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     1741 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     1801 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     1861 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     1921 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     1981 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     2041 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     2101 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     2161 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     2221 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     2281 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     2341 gcgcacattt ccccgaaaag tgccacct
//
