pGL3-DNMT1-U6-Nicking sgRNA

LOCUS      pGL3-DNMT1-U6-Nicking sgRNA             4951 bp DNA     circular SYN 12-MAR-2026
DEFINITION  expresses sgRNA from the U6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4951)
  AUTHORS   Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins 
            A, Iyer V, Huang X, Skarnes WC
  TITLE     Efficient genome modification by CRISPR-Cas9 nickase with minimal 
            off-target effects.
  JOURNAL   Nat Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857.
  PUBMED    24584192
REFERENCE   2  (bases 1 to 4951)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 19, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857."
FEATURES             Location/Qualifiers
     source          1..4951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=OL205
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(322..346)
                     /label=OL211
     misc_feature    322..342
                     /label=DMNT1 nickingRNA
     primer_bind     322..342
                     /label=OL972
     misc_RNA        343..418
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    470..587
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     complement(603..624)
                     /label=OL206
     promoter        636..1146
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1065..1083
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1168..1767
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1168..1187)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1664..1684
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1914..2035
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1925..1944)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1979..1998
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2283..2300)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2454..3042)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2534..2553)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3213..4073)
                     /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     3836..3855
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4074..4178)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4205..4660
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4292..4311)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4502..4523
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4791..4839
                     /note="synthetic polyadenylation signal"
     misc_feature    4853..4944
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4893..4912
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc ggcccttcag ctaaaataaa gggttttaga gctagaaata
      361 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      421 tttttaaaga attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa
      481 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
      541 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
      601 gggacagcag agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa
      661 ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc
      721 ggcgccgacc ctgggactcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc
      781 gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt
      841 tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc
      901 ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc
      961 aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag
     1021 gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc
     1081 aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct
     1141 ccccaggggg atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc
     1201 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     1261 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     1321 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     1381 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     1441 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     1501 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     1561 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     1621 gagcgcgccg gggtgcccgc cttcctggaa acctccgcgc cccgcaacct ccccttctac
     1681 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     1741 tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa
     1801 aggagcgcac gaccccatgc atcggtacct ttaagaccaa tgacttacaa ggcagctgta
     1861 gatcttagcc actttctaga gtcggggcgg ccggccgctt cgagcagaca tgataagata
     1921 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     1981 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     2041 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
     2101 caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccgtcgaccg atgcccttga
     2161 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     2221 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctcttccgct
     2281 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2341 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2401 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2461 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2521 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2581 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2641 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2701 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2761 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2821 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2881 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2941 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3001 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3061 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3121 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3181 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3241 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3301 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgggaccca
     3361 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3421 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3481 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3541 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3601 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3661 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3721 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3781 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3841 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3901 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3961 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4021 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4081 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4141 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4201 cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4261 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4321 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4381 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     4441 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4501 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     4561 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     4621 tttaacgcga attttaacaa aatattaacg tttacaattt cccattcgcc attcaggctg
     4681 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gcccaagcta
     4741 ccatgataag taagtaatat taaggtacgg gaggtacttg gagcggccgc aataaaatat
     4801 ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta ctaacatacg
     4861 ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc cccagtgcaa
     4921 gtgcaggtgc cagaacattt ctctatcgat a
//