LOCUS       sequence-437046-        2334 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hNOLC1 atgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2334)
  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 2334)
  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: pDY2259 hNOLC1 atgRNA Paired Guide 1
FEATURES             Location/Qualifiers
     source          1..2334
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     393..412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(631..735)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        842..1090
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     842..862
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1013..1032
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1111..1186
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1191..1228
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     rep_origin      complement(1345..1933)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1425..1444)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2104..2334,1..561,562..630))
                     /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 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
       61 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      121 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      181 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      241 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      301 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      361 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      421 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      481 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
      541 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
      601 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
      661 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
      721 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgcta gctgtacaaa
      781 aaagcaggct ttaaaggaac caattcagtc gactggatcc ggtaccaagg tcgggcagga
      841 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
      901 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
      961 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     1021 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     1081 acgaaacacc gcgtattgcc tggaggatgg gttttagagc tagaaatagc aagttaaaat
     1141 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcccgg atgatcctga
     1201 cgacggagac cgccgtcgtc gacaagccgg cctcctccag gtttttttaa gcttgggccg
     1261 ctcgaggtac ctctctacat atgacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     1321 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     1381 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1441 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     1501 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     1561 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     1621 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     1681 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     1741 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     1801 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     1861 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     1921 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     1981 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     2041 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     2101 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2161 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2221 ccccagtgct gcaatgatac cgcgagatcc acgctcaccg gctccagatt tatcagcaat
     2281 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgc
//
