LOCUS       sequence-454003-        8303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Nme2Smu Cas9 nuclease with D56R mutation in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8303)
  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 8303)
  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: pEJS2696-CMV-Nme2SmuCas9-D56R
FEATURES             Location/Qualifiers
     source          1..8303
                     /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"
     regulatory      2987..2996
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3449..3469
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3478..3495
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3518..3535)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3524..3748
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3819..3835)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3819..3835)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3832..3854)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3843..3859
                     /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(3867..3873,3874..3891,3892..3897))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3912..3933
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    4050..4171
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4061..4080)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4115..4134
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4329..4727)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(4746..4793)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(4841..5170)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4855..4990
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4909..4928)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5155..5175
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(5294..5481,5482..5493)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     rep_origin      complement(5554..6142)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5634..5653)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6313..7104,7105..7173))
                     /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     6936..6955
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7174..7278)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7357..7376)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    join(7568..7579,7580..7717)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     enhancer        7726..8105
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8106..8303,1..6)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8260..8280
                     /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 gacaacgcca ccatggtgcg
     3001 ggtggacgtg tacaccaagg ccggcaagaa ctacctggtt cctgtgtacg tgtggcaggt
     3061 ggcccagggc atcttaccca accgcgccgt gaccagcggc aagtccgagg ctgactggga
     3121 cctgatcgat gagagcttcg agttcaagtt ctctctgtcc cggggagatc tcgtggaaat
     3181 gatctccaac aagggcagaa tcttcggcta ctacaacggc ctggacagag ccaacggctc
     3241 tattggaatt agagagcacg acctagagaa gagcaagggc aaagacggcg tgcatagagt
     3301 gggagtgaaa acagctacag catttaacaa gtaccacgtg gatcccctgg gcaaagagat
     3361 ccacagatgc agcagcgaac ccagacctac actgaaaatc aagtctaaga aggaggataa
     3421 aagaaccgcc gacggcagcg aattcgagcc caagaagaag aggaaagtct aaccggtcat
     3481 catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     3541 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3601 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3661 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3721 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg
     3781 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     3841 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3901 tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     3961 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4021 ggtttgcgta ttgggcgcta cagacatgat aagatacatt gatgagtttg gacaaaccac
     4081 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4141 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat
     4201 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
     4261 catagaaggc ggcggtggaa tcgaaatctc gtagcacgtg tcagtcctgc tcctcggcca
     4321 cgaagtgctt agccctccca cacataacca gagggcagca attcacgaat cccaactgcc
     4381 gtcggctgtc catcactgtc cttcactatg gctttgatcc caggatgcag atcgagaagc
     4441 acctgtcggc accgtccgca ggggctcaag atgcccctgt tctcatttcc gatcgcgacg
     4501 atacaagtca ggttgccagc tgccgcagca gcagcagtgc ccagcaccac gagttctgca
     4561 caaggtcccc cagtaaaatg atatacattg acaccagtga agatgcggcc gtcgctagag
     4621 agagctgcgc tggcgacgct gtagtcttca gagatgggga tgctgttgat tgtagccgtt
     4681 gctctttcaa tgagggtgga ttcttcttga gacaaaggct tggccatggt ttagttcctc
     4741 accttgtcgt attatactat gccgatatac tatgccgatg attaattgtc aacacgtgct
     4801 gatcagatcc gaaaatggat atacaagctc ccgggagctt tttgcaaaag cctaggcctc
     4861 caaaaaagcc tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg
     4921 cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta
     4981 ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc
     5041 tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat
     5101 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta
     5161 actgacacac attccacaga attcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5221 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5281 ggaaagaaca tgtaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt
     5341 aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg
     5401 atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat
     5461 caagtaaaat tactcaagta ctttacacct ctgcatgtga gcaaaaggcc agcaaaaggc
     5521 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5581 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5641 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5701 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5761 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5821 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5881 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5941 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6001 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6061 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6121 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacactca
     6181 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6241 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6301 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6361 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6421 accatctggc cccagtgctg caatgatacc gcgggaccca cgctcaccgg ctccagattt
     6481 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6541 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6601 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6661 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6721 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6781 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6841 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6901 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6961 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7021 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7081 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7141 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7201 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7261 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     7321 agatcgatct cccgatcccc tagggtcgac tctcagtaca atctgctctg atgccgcata
     7381 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     7441 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     7501 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgggtac cggcatatgg
     7561 ttcttgacag aggtgtaaaa agtactcaaa aattttactc aagtgaaagt acaagtactt
     7621 agggaaaatt ttactcaatt aaaagtaaaa gtatctggct agaatcttac ttgagtaaaa
     7681 gtaaaaaagt actccattaa aattgtactt gagtattaac gcgttgacat tgattattga
     7741 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     7801 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     7861 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     7921 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     7981 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     8041 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     8101 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     8161 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     8221 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     8281 tacggtggga ggtctatata agc
//
