LOCUS       sequence-490930-        8568 bp    DNA     circular SYN 28-OCT-2025
DEFINITION  All-in-one plasmid. Expresses R-NmeCas9 in mammalian cells for RNA 
            knockdown. Spacer targeting EZH2 gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8568)
  AUTHORS   Xu C, Niu X, Sun H, Yan H, Tang W, Ke A
  TITLE     Conversion of IscB and Cas9 into RNA-guided RNA editors.
  JOURNAL   Cell. 2025 Aug 18:S0092-8674(25)00854-2. doi: 
            10.1016/j.cell.2025.07.032.
  PUBMED    40829585
REFERENCE   2  (bases 1 to 8568)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 28, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2025 
            Aug 18:S0092-8674(25)00854-2. doi: 10.1016/j.cell.2025.07.032."
COMMENT     Sequence Label: NLS-R-NmeCas9 (D16A)-HA-NLS_sgRNA EZH guide
FEATURES             Location/Qualifiers
     source          1..8568
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(36..128)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(180..428)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(238..257)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(408..428)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(485..504)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        654..1816
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          885..1807
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1764..1784
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1822..1831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1834..1854
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4786..4812
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4819..4845
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     primer_bind     complement(4861..4878)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4867..5091
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5256..6369
                     /label=mini-oriP
                     /note="internally truncated variant of the Epstein-Barr 
                     virus oriP replication origin (Tanaka et al., 1999)"
     primer_bind     complement(6414..6430)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6414..6430)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6427..6449)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6438..6454
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6462..6468,6469..6486,6487..6492))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6507..6528
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6645..6662)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6816..7404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6896..6915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7575..8366,8367..8435))
                     /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     8198..8217
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8436..8540)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct gtttaaacaa aaaaataaac gatgcccctt aaagcagaag
       61 ctttaagggg cagagcgttg cggcacatct tttcagacgg ccttattgta gcaacggttc
      121 tcatttcgtt tccgaaatga gaaagggagc tacaaccctt gtggaccaca gtgttaccag
      181 gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt tcaagttacg
      241 gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta cccaagaaat
      301 tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt caaattaatt
      361 ctaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat catgggaaat
      421 aggccctctt cctgcccgac cttgacgtcg acagatctcc cgatccccta tggtgcactc
      481 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
      541 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
      601 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc ggaggctccg
      661 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
      721 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
      781 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
      841 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg
      901 gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca
      961 cctggctcca gtacgtgatt cttgatcccg agctggagcc aggggcgggc cttgcgcttt
     1021 aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt
     1081 gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta
     1141 aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg
     1201 ccaggatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc
     1261 gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg
     1321 ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc
     1381 cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc
     1441 gcttcccggc cctgctccag ggggctcaaa atggaggacg cggcgctcgg gagagcgggc
     1501 gggtgagtca cccacacaaa ggaaaggggc ctttccgtcc tcagccgtcg cttcatgtga
     1561 ctccacggag taccgggcgc cgtccaggca cctcgattag ttctggagct tttggagtac
     1621 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     1681 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     1741 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     1801 atttcaggtg tcgtgaacac cgccaccatg gtgcctaaga agaagagaaa ggtggctgcc
     1861 ttcaaaccta attcaatcaa ctacatcctc ggcctcgcta tcggcatcgc atccgtcggc
     1921 tgggcgatgg tagaaattga cgaagaagaa aaccccatcc gcctgattga tttgggcgtg
     1981 cgcgtatttg agcgtgccga agtaccgaaa acaggcgact cccttgccat ggcaaggcgt
     2041 ttggcgcgca gtgttcgccg cctgacccgc cgtcgcgccc accgcctgct tcggacccgc
     2101 cgcctattga aacgcgaagg cgtattacaa gccgccaatt ttgacgaaaa cggcttgatt
     2161 aaatccttac cgaatacacc atggcaactt cgcgcagccg cattagaccg caaactgacg
     2221 cctttagagt ggtcggcagt cttgttgcat ttaatcaaac atcgcggcta tttatcgcaa
     2281 cggaaaaacg agggcgaaac tgccgataag gagcttggcg ctttgcttaa aggcgtagcc
     2341 ggcaatgccc atgccttaca gacaggcgat ttccgcacac cggccgaatt ggctttaaat
     2401 aaatttgaga aagaaagcgg ccatatccgc aatcagcgca gcgattattc gcatacgttc
     2461 agccgcaaag atttacaggc ggagctgatt ttgctgtttg aaaaacaaaa agaatttggc
     2521 aatccgcatg tttcaggcgg ccttaaagaa ggtattgaaa ccctactgat gacgcaacgc
     2581 cctgccctgt ccggcgatgc cgttcaaaaa atgttggggc attgcacctt cgaaccggca
     2641 gagccgaaag ccgctaaaaa cacctacaca gccgaacgtt tcatctggct gaccaagctg
     2701 aacaacctgc gtattttaga gcaaggcagc gagcggccat tgaccgatac cgaacgcgcc
     2761 acgcttatgg acgagccata cagaaaatcc aaactgactt acgcacaagc ccgtaagctg
     2821 ctgggtttag aagataccgc ctttttcaaa ggcttgcgct atggtaaaga caatgccgaa
     2881 gcctcaacat tgatggaaat gaaggcctac catgccatca gccgtgcact ggaaaaagaa
     2941 ggattgaaag acaaaaaatc cccattaaac ctttctcccg aattacaaga cgaaatcggc
     3001 acggcattct ccctgttcaa aaccgatgaa gacattacag gccgtctgaa agaccgtata
     3061 cagcccgaaa tcttagaagc gctgttgaaa cacatcagct tcgataagtt cgtccaaatt
     3121 tccttgaaag cattgcgccg aattgtgcct ctaatggaac aaggcaaacg ttacgatgaa
     3181 gcctgcgccg aaatctacgg agaccattac ggcaagaaga atacggaaga aaagatttat
     3241 ctgccgccga ttcccgccga cgaaatccgc aaccccgtcg tcttgcgcgc cttatctcaa
     3301 gcacgtaagg tcattaacgg cgtggtacgc cgttacggct ccccagctcg tatccatatt
     3361 gaaactgcaa gggaagtagg taaatcgttt aaagaccgca aagaaattga gaaacgccaa
     3421 gaagaaaacc gcaaagaccg ggaaaaagcc gccgccaaat tccgagagta tttccccaat
     3481 tttgtcggag aacccaaatc caaagatatt ctgaaactgc gcctgtacga gcaacaacac
     3541 ggcaaatgcc tgtattcggg caaagaaatc aacttaggcc gtctgaacga aaaaggctat
     3601 gtcgaaatcg accatgccct gccgttctcg cgcacatggg acgacagttt caacaataaa
     3661 gtactggtat tgggcagcga aaaccaaaac aaaggcaatc aaacccctta cgaatacttc
     3721 aacggcaaag acaacagccg cgaatggcag gaatttaaag cgcgtgtcga aaccagccgt
     3781 ttcccgcgca gtaaaaaaca acggattctg ctgcaaaaat tcgatgaaga cggctttaaa
     3841 gaacgcaatc tgaacgacac gcgctacgtc aaccgtttcc tgtgtcaatt tgttgccgac
     3901 cgtatgcggc tgacaggtaa aggcaagaaa cgtgtctttg catccaacgg acaaattacc
     3961 aatctgttgc gcggcttttg gggattgcgc aaagtgcgtg cggaaaacga ccgccatcac
     4021 gccttggacg ccgtcgtcgt tgcctgctcg accgttgcca tgcagcagaa aattacccgt
     4081 tttgtacgct ataaagagat gaacgcgttt gacggtaaaa ccatagacaa agaaacagga
     4141 gaagtgctgc atcaaaaaac acacttccca caaccttggg aatttttcgc acaagaagtc
     4201 atgattcgcg tcttcggcaa accggacggc aaacccgaat tcgaagaagc cgatacccta
     4261 gaaaaactgc gcacgttgct tgccgaaaaa ttatcatctc gccccgaagc cgtacacgaa
     4321 tacgttacgc cactgtttgt ttcacgcgcg cccaatcgga agatgagcgg gcaagggcat
     4381 atggagaccg tcaaatccgc caaacgactg gacgaaggcg tcagcgtgtt gcgcgtaccg
     4441 ctgacacagt taaaactgaa agacttggaa aaaatggtca atcgggagcg cgaacctaag
     4501 ctatacgaag cactgaaagc acggctggaa gcacataaag acgatcctgc caaagccttt
     4561 gccgagccgt tttacaaata cgataaagca ggcaaccgca cccaacaggt aaaagccgta
     4621 cgcgtagagc aagtacagaa aaccggcgta tgggtgcgca accataacgg tattgccgac
     4681 aacgcaacca tggtgcgcgt acaccctaat gattacaaat accaaattga cgaactgggc
     4741 aaagaaatca gaccatgccg tctgaaaaaa cgcccgcctg tccgttaccc atacgatgtt
     4801 ccagattacg ctgcagctcc agcagcgaag aaaaagaagc tggattaact cgctgatcag
     4861 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     4921 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     4981 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     5041 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     5101 cggaaagaac cagctggggc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     5161 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     5221 caactcgtag gacaggtgcc ctggccgggg tcccgcagga aaaggacaag cagcgaaaat
     5281 tcacgccccc ttgggaggtg gcggcatatg caaaggatag cactcccact ctactactgg
     5341 gtatcatatg ctgactgtat atgcatgagg atagcatatg ctacccggat acagattagg
     5401 atagcatata ctacccagat atagattagg atagcatatg ctacccagat atagattagg
     5461 atagcctatg ctacccagat ataaattagg atagcatata ctacccagat atagattagg
     5521 atagcatatg ctacccagat atagattagg atagcctatg ctacccagat atagattagg
     5581 atagcatatg ctacccagat atagattagg atagcatatg ctatccagat atttgggtag
     5641 tatatgctac ccagatataa attaggatag catatactac cctaatctct attaggatag
     5701 catatgctac ccggatacag attaggatag catatactac ccagatatag attaggatag
     5761 catatgctac ccagatatag attaggatag cctatgctac ccagatataa attaggatag
     5821 catatactac ccagatatag attaggatag catatgctac ccagatatag attaggatag
     5881 cctatgctac ccagatatag attaggatag catatgctat ccagatattt gggtagtata
     5941 tgctatccat ggcaacatta gcccaccgtg ctctcagcga cctcgtgaat atgaggacca
     6001 acaaccctgt gcttggcgct caggcgcaag tgtgtgtaat ttgtcctcca gatcgcagca
     6061 atcgcgcccc tatcttggcc cgcccaccta cttatgcagg tattccccgg ggtgccatta
     6121 gtggttttgt gggcaagtgg tttgaccgca gtggttagcg gggttacaat cagccaagtt
     6181 attacaccct tattttacag tccaaaaccg cagggcggcg tgtgggggct gacgcgatcg
     6241 ctgttcctta ggaccctttt actaacccta attcgatagc atatgcttcc cgttgggtaa
     6301 catatgctat tgaattaggg ttagtctgga tagtatatac tactacccgg gaagcatatg
     6361 ctacccgttt agggtttacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     6421 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     6481 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     6541 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6601 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     6661 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     6721 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     6781 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     6841 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     6901 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     6961 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     7021 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     7081 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     7141 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     7201 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     7261 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     7321 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7381 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     7441 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     7501 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     7561 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     7621 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7681 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7741 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     7801 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     7861 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     7921 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     7981 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     8041 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     8101 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     8161 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     8221 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     8281 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     8341 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     8401 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     8461 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     8521 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgac
//
