LOCUS       sequence-356969-        2329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To insert attB attachment site at CCR5 in human cells via twinPE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2329)
  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 2329)
  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-CCR5_DF_A509b_attB
FEATURES             Location/Qualifiers
     source          1..2329
                     /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(580..1168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(660..679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1339..2130,2131..2199))
                     /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     1962..1981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2200..2304)
                     /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 gaagtgtgat cacttgggtg ggttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      421 tcggtgcatg atcctgacga cggagaccgc cgtcgtcgac aagcccccaa gtgatctttt
      481 tttaagcttg ggccgctcga ggtacctctc tacatatgac atgtgagcaa aaggccagca
      541 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      601 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      661 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      721 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      781 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      841 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      901 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      961 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     1021 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     1081 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1141 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     1201 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1261 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1321 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1381 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1441 gggcttacca tctggcccca gtgctgcaat gataccgcga gatccacgct caccggctcc
     1501 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1561 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1621 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1681 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     1741 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     1801 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     1861 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     1921 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     1981 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     2041 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     2101 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     2161 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     2221 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2281 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacct
//
