LOCUS       sequence-464563-        2367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  attachment site pegRNA for human NOLC1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2367)
  AUTHORS   Fell CW, Schmitt-Ulms C, Tagliaferri DV, Gootenberg JS, Abudayyeh OO
  TITLE     Precise kilobase-scale genomic insertions in mammalian cells using 
            PASTE.
  JOURNAL   Nat Protoc. 2024 Dec 15. doi: 10.1038/s41596-024-01090-z.
  PUBMED    39676077
REFERENCE   2  (bases 1 to 2367)
  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 Protoc.
            2024 Dec 15. doi: 10.1038/s41596-024-01090-z."
COMMENT     Sequence Label: NOLC1 atgRNA pDY2250
FEATURES             Location/Qualifiers
     source          1..2367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     189..208
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(427..531)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        638..886
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     638..658
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     809..828
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        907..982
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1016..1053
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     rep_origin      complement(1174..1762)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1254..1273)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1933..2367,1..357,358..426))
                     /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"
ORIGIN
        1 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
       61 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
      121 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
      181 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
      241 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
      301 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
      361 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
      421 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
      481 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
      541 ccgaaaagtg ccacctgacg tcgctagctg tacaaaaaag caggctttaa aggaaccaat
      601 tcagtcgact ggatccggta ccaaggtcgg gcaggaagag ggcctatttc ccatgattcc
      661 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
      721 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
      781 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
      841 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcgt attgcctgga
      901 ggatgggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      961 aaaagtggca ccgagtcggt gcgaaccacg cggcgaatgc cggcgtccgc cccggatgat
     1021 cctgacgacg gagaccgccg tcgtcgacaa gccggcctcc tccaggcaat tttttttaag
     1081 cttgggccgc tcgaggtacc tctctacata tgacatgtga gcaaaaggcc agcaaaaggc
     1141 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1201 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1261 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1321 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1381 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1441 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1501 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1561 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     1621 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1681 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1741 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1801 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     1861 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     1921 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     1981 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2041 accatctggc cccagtgctg caatgatacc gcgagatcca cgctcaccgg ctccagattt
     2101 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2161 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2221 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2281 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2341 gtgcaaaaaa gcggttagct ccttcgg
//
