LOCUS       sequence-453991-        8279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Nme2 Cas9 nuclease with D56R mutation in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8279)
  AUTHORS   Bamidele N, Ansodaria A, Chen Z, Cheng H, Panwala R, Jazbec E, 
            Sontheimer EJ
  TITLE     Rational Design of Enhanced Nme2Cas9 and Nme2SmuCas9 Nucleases and 
            Base Editors
  JOURNAL   bioRxiv November 07, 2024
REFERENCE   2  (bases 1 to 8279)
  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: "bioRxiv 
            November 07, 2024"
COMMENT     Sequence Label: pEJS2698-CMV-Nme2Cas9-D56R
FEATURES             Location/Qualifiers
     source          1..8279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..67
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        48..66
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             116..136
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3425..3445
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3454..3471
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3494..3511)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3500..3724
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3795..3811)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3795..3811)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3808..3830)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3819..3835
                     /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(3843..3849,3850..3867,3868..3873))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3888..3909
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    4026..4147
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4037..4056)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4091..4110
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4305..4703)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(4722..4769)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(4817..5146)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4831..4966
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4885..4904)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5131..5151
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(5270..5457,5458..5469)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     rep_origin      complement(5530..6118)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5610..5629)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6289..7080,7081..7149))
                     /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     6912..6931
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7150..7254)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7333..7352)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    join(7544..7555,7556..7693)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     enhancer        7702..8081
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8082..8279,1..6)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8236..8256
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 agagctggtt tagtgaaccg tcagatccgc tagagatccg cggccgctaa tacgactcac
       61 tatagggaga gccgccacca tgaaacggac agccgacgga agcgagttcg agtcaccaaa
      121 gaagaagcgg aaagtcgaag atatggccgc cttcaagcct aacccaatca attacatcct
      181 gggactggac atcggaatcg catccgtggg atgggctatg gtggagatcg acgaggagga
      241 gaatcctatc cggctgatcg atctgggcgt gagagtgttt gagagggccg aggtgccaaa
      301 gaccggccgt tctctggcta tggcccggag actggcacgg agcgtgaggc gcctgacacg
      361 gagaagggca cacaggctgc tgagggcacg ccggctgctg aagagagagg gcgtgctgca
      421 ggcagcagac ttcgatgaga atggcctgat caagagcctg ccaaacaccc cctggcagct
      481 gagagcagcc gccctggaca ggaagctgac accactggag tggtctgccg tgctgctgca
      541 cctgatcaag caccgcggct acctgagcca gcggaagaac gagggagaga cagcagacaa
      601 ggagctgggc gccctgctga agggagtggc caacaatgcc cacgccctgc agaccggcga
      661 tttcaggaca cctgccgagc tggccctgaa taagtttgag aaggagtccg gccacatcag
      721 aaaccagagg ggcgactata gccacacctt ctcccgcaag gatctgcagg ccgagctgat
      781 cctgctgttc gagaagcaga aggagtttgg caatccacac gtgagcggag gcctgaagga
      841 gggaatcgag accctgctga tgacacagag gcctgccctg tccggcgacg cagtgcagaa
      901 gatgctggga cactgcacct tcgagcctgc agagccaaag gccgccaaga acacctacac
      961 agccgagcgg tttatctggc tgacaaagct gaacaatctg agaatcctgg agcagggatc
     1021 cgagaggcca ctgaccgaca cagagagggc caccctgatg gatgagcctt accggaagtc
     1081 taagctgaca tatgcccagg ccagaaagct gctgggcctg gaggacaccg ccttctttaa
     1141 gggcctgaga tacggcaagg ataatgccga ggcctccaca ctgatggaga tgaaggccta
     1201 tcacgccatc tctcgcgccc tggagaagga gggcctgaag gacaagaagt cccccctgaa
     1261 cctgagctcc gagctgcagg atgagatcgg caccgccttc tctctgttta agaccgacga
     1321 ggatatcaca ggccgcctga aggacagggt gcagcctgag atcctggagg ccctgctgaa
     1381 gcacatctct ttcgataagt ttgtgcagat cagcctgaag gccctgagaa ggatcgtgcc
     1441 actgatggag cagggcaagc ggtacgacga ggcctgcgcc gagatctacg gcgatcacta
     1501 tggcaagaag aacacagagg agaagatcta tctgccccct atccctgccg acgagatcag
     1561 aaatcctgtg gtgctgaggg ccctgtccca ggcaagaaaa gtgatcaacg gagtggtgcg
     1621 ccggtacgga tctccagccc ggatccacat cgagaccgcc agagaagtgg gcaagagctt
     1681 caaggaccgg aaggagatcg agaagagaca ggaggagaat cgcaaggatc gggagaaggc
     1741 cgccgccaag tttagggagt acttccctaa ctttgtgggc gagccaaagt ctaaggacat
     1801 cctgaagctg cgcctgtacg agcagcagca cggcaagtgt ctgtatagcg gcaaggagat
     1861 caatctggtg cggctgaacg agaagggcta tgtggagatc gatcacgccc tgcctttctc
     1921 cagaacctgg gacgattctt ttaacaataa ggtgctggtg ctgggcagcg agaaccagaa
     1981 taagggcaat cagacaccat acgagtattt caatggcaag gacaactcca gggagtggca
     2041 ggagttcaag gcccgcgtgg agacctctag atttcccagg agcaagaagc agcggatcct
     2101 gctgcagaag ttcgacgagg atggctttaa ggagtgcaac ctgaatgaca ccagatacgt
     2161 gaaccggttc ctgtgccagt ttgtggccga tcacatcctg ctgaccggca agggcaagag
     2221 aagggtgttc gcctctaatg gccagatcac aaacctgctg aggggatttt ggggactgag
     2281 gaaggtgcgg gcagagaatg acagacacca cgcactggat gcagtggtgg tggcatgcag
     2341 caccgtggca atgcagcaga agatcacaag attcgtgagg tataaggaga tgaacgcctt
     2401 tgacggcaag accatcgata aggagacagg caaggtgctg caccagaaga cccacttccc
     2461 ccagccttgg gagttctttg cccaggaagt gatgatccgg gtgttcggca agccagacgg
     2521 caagcctgag tttgaggagg ccgatacccc agagaagctg aggacactgc tggcagagaa
     2581 gctgtctagc aggccagagg cagtgcacga gtacgtgacc ccactgttcg tgtccagggc
     2641 acccaatcgg aagatgtctg gcgcccacaa ggacacactg agaagcgcca agaggtttgt
     2701 gaagcacaac gagaagatct ccgtgaagag agtgtggctg accgagatca agctggccga
     2761 tctggagaac atggtgaatt acaagaacgg cagggagatc gagctgtatg aggccctgaa
     2821 ggcaaggctg gaggcctacg gaggaaatgc caagcaggcc ttcgacccaa aggataaccc
     2881 cttttataag aagggaggac agctggtgaa ggccgtgcgg gtggagaaga cccaggagag
     2941 cggcgtgctg ctgaataaga agaacgccta cacaatcgcc gacaatggcg atatggtgag
     3001 agtggacgtg ttctgtaagg tggataagaa gggcaagaat cagtacttta tcgtgcctat
     3061 ctatgcctgg caggtggccg agaacatcct gccagacatc gattgcaagg gctacagaat
     3121 cgacgatagc tatacattct gtttttccct gcacaagtat gacctgatcg ccttccagaa
     3181 ggatgagaag tccaaggtgg agtttgccta ctatatcaat tgcgactcct ctaacggcag
     3241 gttctacctg gcctggcacg ataagggcag caaggagcag cagtttcgca tctccaccca
     3301 gaatctggtg ctgatccaga agtatcaggt gaacgagctg ggcaaggaga tcaggccatg
     3361 tcggctgaag aagcgcccac ccgtgcggga ggataaaaga accgccgacg gcagcgaatt
     3421 cgagcccaag aagaagagga aagtctaacc ggtcatcatc accatcacca ttgagtttaa
     3481 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     3541 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     3601 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3661 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3721 atggcttctg aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc
     3781 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3841 cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa
     3901 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3961 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctacaga
     4021 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     4081 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     4141 acaagttggg gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc
     4201 cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga
     4261 aatctcgtag cacgtgtcag tcctgctcct cggccacgaa gtgcttagcc ctcccacaca
     4321 taaccagagg gcagcaattc acgaatccca actgccgtcg gctgtccatc actgtccttc
     4381 actatggctt tgatcccagg atgcagatcg agaagcacct gtcggcaccg tccgcagggg
     4441 ctcaagatgc ccctgttctc atttccgatc gcgacgatac aagtcaggtt gccagctgcc
     4501 gcagcagcag cagtgcccag caccacgagt tctgcacaag gtcccccagt aaaatgatat
     4561 acattgacac cagtgaagat gcggccgtcg ctagagagag ctgcgctggc gacgctgtag
     4621 tcttcagaga tggggatgct gttgattgta gccgttgctc tttcaatgag ggtggattct
     4681 tcttgagaca aaggcttggc catggtttag ttcctcacct tgtcgtatta tactatgccg
     4741 atatactatg ccgatgatta attgtcaaca cgtgctgatc agatccgaaa atggatatac
     4801 aagctcccgg gagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct
     4861 ggaatagctc agaggcagag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     4921 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg
     4981 gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     5041 agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc
     5101 tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc cacagaattc
     5161 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5221 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgta agtgatctcc
     5281 aaaaaataag tactttttga ctgtaaataa aattgtaagg agtaaaaagt actttttttt
     5341 ctaaaaaaat gtaattaagt aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag
     5401 taaaaatccc caaaaataat acttaagtac agtaatcaag taaaattact caagtacttt
     5461 acacctctgc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5521 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5581 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5641 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5701 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5761 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5821 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5881 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5941 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6001 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6061 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6121 agatcctttg atcttttcta cggggtctga cactcagtgg aacgaaaact cacgttaagg
     6181 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6241 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6301 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6361 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6421 gataccgcgg gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6481 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6541 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6601 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6661 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6721 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6781 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6841 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6901 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6961 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7021 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7081 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7141 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7201 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7261 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg atcccctagg
     7321 gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     7381 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     7441 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     7501 tgtacgggcc agatatacgc gggtaccggc atatggttct tgacagaggt gtaaaaagta
     7561 ctcaaaaatt ttactcaagt gaaagtacaa gtacttaggg aaaattttac tcaattaaaa
     7621 gtaaaagtat ctggctagaa tcttacttga gtaaaagtaa aaaagtactc cattaaaatt
     7681 gtacttgagt attaacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     7741 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     7801 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     7861 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     7921 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     7981 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     8041 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     8101 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     8161 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     8221 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagc
//
