LOCUS       sequence-355667-        2328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To insert attB-rev attachment site at IDS in human cells via twinPE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2328)
  AUTHORS   Anzalone AV, Gao XD, Podracky CJ, Nelson AT, Koblan LW, Raguram A, 
            Levy JM, Mercer JAM, Liu DR
  TITLE     Programmable deletion, replacement, integration and inversion of 
            large DNA sequences with twin prime editing.
  JOURNAL   Nat Biotechnol. 2022 May;40(5):731-740. doi: 
            10.1038/s41587-021-01133-w. Epub 2021 Dec 9.
  PUBMED    34887556
REFERENCE   2  (bases 1 to 2328)
  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/s41587-021-01133-w"; journalName: "Nat Biotechnol"; date: 
            "2022-05"; volume: "40"; issue: "5"; pages: "731-740"
COMMENT     Sequence Label: pU6-Sp-gRNA-IDS_DF_D1b_attB_rev
FEATURES             Location/Qualifiers
     source          1..2328
                     /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"
     protein_bind    428..465
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     rep_origin      complement(579..1167)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(659..678)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1338..2129,2130..2198))
                     /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     1961..1980
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2199..2303)
                     /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 gctgtggaac tgcaacacac tgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      421 tcggtgcggc ttgtcgacga cggcggtctc cgtcgtcagg atcatgtgtt gcagtttttt
      481 ttaagcttgg gccgctcgag gtacctctct acatatgaca tgtgagcaaa aggccagcaa
      541 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
      601 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
      661 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
      721 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
      781 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
      841 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
      901 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
      961 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     1021 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     1081 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     1141 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     1201 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     1261 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     1321 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     1381 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     1441 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     1501 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     1561 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     1621 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     1681 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     1741 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     1801 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     1861 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     1921 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     1981 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     2041 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     2101 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     2161 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     2221 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     2281 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacct
//
