LOCUS       sequence-355650-        2331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To insert attB attachment site at ALB in human cells via twinPE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2331)
  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 2331)
  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-ALB_DF_B358c_attB
FEATURES             Location/Qualifiers
     source          1..2331
                     /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(582..1170)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(662..681)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1341..2132,2133..2201))
                     /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     1964..1983
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2202..2306)
                     /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 gatttatgag atcaacagca cgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      421 tcggtgcggc ttgtcgacga cggcggtctc cgtcgtcagg atcatctgtt gatctcattt
      481 tttttaagct tgggccgctc gaggtacctc tctacatatg acatgtgagc aaaaggccag
      541 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      601 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      661 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      721 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      781 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      841 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      901 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      961 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     1021 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1081 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1141 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1201 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     1261 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     1321 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     1381 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     1441 gagggcttac catctggccc cagtgctgca atgataccgc gagatccacg ctcaccggct
     1501 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     1561 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     1621 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     1681 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     1741 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     1801 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     1861 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     1921 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     1981 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     2041 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     2101 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     2161 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     2221 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2281 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc t
//
