LOCUS       sequence-454017-        8927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminal ABE8e-Nme2Smu nCas9 (D16A) base editor with 
            E932R mutation in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8927)
  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 8927)
  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: pEJS2701-CMV-Nme2Smu-ABE8e-nt-E932R
FEATURES             Location/Qualifiers
     source          1..8927
                     /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      3611..3620
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4073..4093
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4102..4119
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4142..4159)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4148..4372
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4443..4459)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4443..4459)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4456..4478)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4467..4483
                     /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(4491..4497,4498..4515,4516..4521))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4536..4557
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    4674..4795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4685..4704)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4739..4758
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4953..5351)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(5370..5417)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5465..5794)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5479..5614
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5533..5552)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5779..5799
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(5918..6105,6106..6117)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     rep_origin      complement(6178..6766)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6258..6277)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6937..7728,7729..7797))
                     /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     7560..7579
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7798..7902)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7981..8000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    join(8192..8203,8204..8341)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     enhancer        8350..8729
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8730..8927,1..6)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8884..8904
                     /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 aaagtcggcg gtagcggcgg aggcagcggt ggcggcagcg gctctgaggt
      181 ggagttttcc cacgagtact ggatgagaca tgccctgacc ctggccaaga gggcacgcga
      241 tgagagggag gtgcctgtgg gagccgtgct ggtgctgaac aatagagtga tcggcgaggg
      301 ctggaacaga gccatcggcc tgcacgaccc aacagcccat gccgaaatta tggccctgag
      361 acagggcggc ctggtcatgc agaactacag actgattgac gccaccctgt acgtgacatt
      421 cgagccttgc gtgatgtgcg ccggcgccat gatccactct aggatcggcc gcgtggtgtt
      481 tggcgtgagg aacagcaaac ggggcgccgc aggctccctg atgaacgtgc tgaactaccc
      541 cggcatgaat caccgcgtcg aaattaccga gggaatcctg gcagatgaat gtgccgccct
      601 gctgtgcgac ttctaccgga tgcctagaca ggtgttcaat gctcagaaga aggcccagag
      661 ctccatcaac tccggaggat ctagcggagg ctcctctggc tctgagacac ctggcacaag
      721 cgagagcgca acacctgaaa gcagcggggg cagcagcggg gggtcaatgg ccgccttcaa
      781 gcctaaccca atcaattaca tcctgggact ggccatcgga atcgcatccg tgggatgggc
      841 tatggtggag atcgacgagg aggagaatcc tatccggctg atcgatctgg gcgtgagagt
      901 gtttgagagg gccgaggtgc caaagaccgg cgattctctg gctatggccc ggagactggc
      961 acggagcgtg aggcgcctga cacggagaag ggcacacagg ctgctgaggg cacgccggct
     1021 gctgaagaga gagggcgtgc tgcaggcagc agacttcgat gagaatggcc tgatcaagag
     1081 cctgccaaac accccctggc agctgagagc agccgccctg gacaggaagc tgacaccact
     1141 ggagtggtct gccgtgctgc tgcacctgat caagcaccgc ggctacctga gccagcggaa
     1201 gaacgaggga gagacagcag acaaggagct gggcgccctg ctgaagggag tggccaacaa
     1261 tgcccacgcc ctgcagaccg gcgatttcag gacacctgcc gagctggccc tgaataagtt
     1321 tgagaaggag tccggccaca tcagaaacca gaggggcgac tatagccaca ccttctcccg
     1381 caaggatctg caggccgagc tgatcctgct gttcgagaag cagaaggagt ttggcaatcc
     1441 acacgtgagc ggaggcctga aggagggaat cgagaccctg ctgatgacac agaggcctgc
     1501 cctgtccggc gacgcagtgc agaagatgct gggacactgc accttcgagc ctgcagagcc
     1561 aaaggccgcc aagaacacct acacagccga gcggtttatc tggctgacaa agctgaacaa
     1621 tctgagaatc ctggagcagg gatccgagag gccactgacc gacacagaga gggccaccct
     1681 gatggatgag ccttaccgga agtctaagct gacatatgcc caggccagaa agctgctggg
     1741 cctggaggac accgccttct ttaagggcct gagatacggc aaggataatg ccgaggcctc
     1801 cacactgatg gagatgaagg cctatcacgc catctctcgc gccctggaga aggagggcct
     1861 gaaggacaag aagtcccccc tgaacctgag ctccgagctg caggatgaga tcggcaccgc
     1921 cttctctctg tttaagaccg acgaggatat cacaggccgc ctgaaggaca gggtgcagcc
     1981 tgagatcctg gaggccctgc tgaagcacat ctctttcgat aagtttgtgc agatcagcct
     2041 gaaggccctg agaaggatcg tgccactgat ggagcagggc aagcggtacg acgaggcctg
     2101 cgccgagatc tacggcgatc actatggcaa gaagaacaca gaggagaaga tctatctgcc
     2161 ccctatccct gccgacgaga tcagaaatcc tgtggtgctg agggccctgt cccaggcaag
     2221 aaaagtgatc aacggagtgg tgcgccggta cggatctcca gcccggatcc acatcgagac
     2281 cgccagagaa gtgggcaaga gcttcaagga ccggaaggag atcgagaaga gacaggagga
     2341 gaatcgcaag gatcgggaga aggccgccgc caagtttagg gagtacttcc ctaactttgt
     2401 gggcgagcca aagtctaagg acatcctgaa gctgcgcctg tacgagcagc agcacggcaa
     2461 gtgtctgtat agcggcaagg agatcaatct ggtgcggctg aacgagaagg gctatgtgga
     2521 gatcgatcac gccctgcctt tctccagaac ctgggacgat tcttttaaca ataaggtgct
     2581 ggtgctgggc agcgagaacc agaataaggg caatcagaca ccatacgagt atttcaatgg
     2641 caaggacaac tccagggagt ggcaggagtt caaggcccgc gtggagacct ctagatttcc
     2701 caggagcaag aagcagcgga tcctgctgca gaagttcgac gaggatggct ttaaggagtg
     2761 caacctgaat gacaccagat acgtgaaccg gttcctgtgc cagtttgtgg ccgatcacat
     2821 cctgctgacc ggcaagggca agagaagggt gttcgcctct aatggccaga tcacaaacct
     2881 gctgagggga ttttggggac tgaggaaggt gcgggcagag aatgacagac accacgcact
     2941 ggatgcagtg gtggtggcat gcagcaccgt ggcaatgcag cagaagatca caagattcgt
     3001 gaggtataag gagatgaacg cctttgacgg caagaccatc gataaggaga caggcaaggt
     3061 gctgcaccag aagacccact tcccccagcc ttgggagttc tttgcccagg aagtgatgat
     3121 ccgggtgttc ggcaagccag acggcaagcc tgagtttgag gaggccgata ccccagagaa
     3181 gctgaggaca ctgctggcag agaagctgtc tagcaggcca gaggcagtgc acgagtacgt
     3241 gaccccactg ttcgtgtcca gggcacccaa tcggaagatg tctggcgccc acaaggacac
     3301 actgagaagc gccaagaggt ttgtgaagca caacgagaag atctccgtga agagagtgtg
     3361 gctgaccgag atcaagctgg ccgatctgga gaacatggtg aattacaaga acggcaggga
     3421 gatcgagctg tatgaggccc tgaaggcaag gctggaggcc tacggaggaa atgccaagca
     3481 ggccttcgac ccaaaggata acccctttta taagaaggga ggacagctgg tgaaggccgt
     3541 gcgggtggag aagacccagc gtagcggcgt gctgctgaat aagaagaacg cctacacaat
     3601 cgccgacaac gccaccatgg tgcgggtgga cgtgtacacc aaggccggca agaactacct
     3661 ggttcctgtg tacgtgtggc aggtggccca gggcatctta cccaaccgcg ccgtgaccag
     3721 cggcaagtcc gaggctgact gggacctgat cgatgagagc ttcgagttca agttctctct
     3781 gtcccgggga gatctcgtgg aaatgatctc caacaagggc agaatcttcg gctactacaa
     3841 cggcctggac agagccaacg gctctattgg aattagagag cacgacctag agaagagcaa
     3901 gggcaaagac ggcgtgcata gagtgggagt gaaaacagct acagcattta acaagtacca
     3961 cgtggatccc ctgggcaaag agatccacag atgcagcagc gaacccagac ctacactgaa
     4021 aatcaagtct aagaaggagg ataaaagaac cgccgacggc agcgaattcg agcccaagaa
     4081 gaagaggaaa gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca
     4141 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4201 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4261 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4321 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     4381 gcggaaagaa ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca
     4441 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4501 gccggaagca taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt
     4561 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4621 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctacagaca tgataagata
     4681 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     4741 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttggggt
     4801 gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga
     4861 aaacgattcc gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtagca
     4921 cgtgtcagtc ctgctcctcg gccacgaagt gcttagccct cccacacata accagagggc
     4981 agcaattcac gaatcccaac tgccgtcggc tgtccatcac tgtccttcac tatggctttg
     5041 atcccaggat gcagatcgag aagcacctgt cggcaccgtc cgcaggggct caagatgccc
     5101 ctgttctcat ttccgatcgc gacgatacaa gtcaggttgc cagctgccgc agcagcagca
     5161 gtgcccagca ccacgagttc tgcacaaggt cccccagtaa aatgatatac attgacacca
     5221 gtgaagatgc ggccgtcgct agagagagct gcgctggcga cgctgtagtc ttcagagatg
     5281 gggatgctgt tgattgtagc cgttgctctt tcaatgaggg tggattcttc ttgagacaaa
     5341 ggcttggcca tggtttagtt cctcaccttg tcgtattata ctatgccgat atactatgcc
     5401 gatgattaat tgtcaacacg tgctgatcag atccgaaaat ggatatacaa gctcccggga
     5461 gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag
     5521 aggcagaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tggggcggag
     5581 aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc gggactatgg
     5641 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     5701 ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     5761 agcctgggga ctttccacac cctaactgac acacattcca cagaattcac tgactcgctg
     5821 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5881 tccacagaat caggggataa cgcaggaaag aacatgtaag tgatctccaa aaaataagta
     5941 ctttttgact gtaaataaaa ttgtaaggag taaaaagtac ttttttttct aaaaaaatgt
     6001 aattaagtaa aagtaaaagt attgattttt aattgtactc aagtaaagta aaaatcccca
     6061 aaaataatac ttaagtacag taatcaagta aaattactca agtactttac acctctgcat
     6121 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6181 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6241 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6301 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6361 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6421 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6481 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6541 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6601 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6661 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6721 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6781 cttttctacg gggtctgaca ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6841 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6901 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6961 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7021 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggga
     7081 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     7141 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     7201 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     7261 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7321 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7381 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7441 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7501 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7561 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7621 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7681 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7741 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7801 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7861 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7921 gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt cgactctcag
     7981 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     8041 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     8101 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     8161 atatacgcgg gtaccggcat atggttcttg acagaggtgt aaaaagtact caaaaatttt
     8221 actcaagtga aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct
     8281 ggctagaatc ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat
     8341 taacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     8401 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     8461 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     8521 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     8581 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     8641 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     8701 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     8761 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     8821 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     8881 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagc
//
