LOCUS       sequence-464560-        2281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nicking sgRNA for NOLC1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2281)
  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 2281)
  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 nicking guide pDY2251
FEATURES             Location/Qualifiers
     source          1..2281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     292..311
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(495..570)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(593..841)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(651..670)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(821..841)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        948..1052
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1053..1121,1122..1913)
                     /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     complement(1271..1290)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(2084..2281,1..391)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
       61 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
      121 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
      181 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
      241 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
      301 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
      361 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
      421 cctggccttt tgctggcctt ttgctcacat gtcatatgta gagaggtacc tcgagcggcc
      481 caagcttaaa aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt
      541 attttaactt gctatttcta gctctaaaac gtatcccctc gtgctcggct ccggtgtttc
      601 gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca
      661 tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact
      721 ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta attctaatta
      781 tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct
      841 cttcctgccc gaccttggta ccggatccag tcgactgaat tggttccttt aaagcctgct
      901 tttttgtaca gctagcgacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      961 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1021 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1081 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1141 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1201 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1261 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1321 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1381 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1441 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1501 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1561 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     1621 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1681 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1741 ctgataaatc tggagccggt gagcgtggat ctcgcggtat cattgcagca ctggggccag
     1801 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     1861 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     1921 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1981 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2041 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2101 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2161 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2221 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2281 c
//
