LOCUS       sequence-454005-        8903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminal ABE8e-Nme2 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 8903)
  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 8903)
  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: pEJS2700-CMV-Nme2-ABE8e-nt-E932R
FEATURES             Location/Qualifiers
     source          1..8903
                     /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             4049..4069
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4078..4095
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4118..4135)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4124..4348
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4419..4435)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4419..4435)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4432..4454)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4443..4459
                     /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(4467..4473,4474..4491,4492..4497))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4512..4533
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    4650..4771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4661..4680)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4715..4734
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4929..5327)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(5346..5393)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5441..5770)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5455..5590
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5509..5528)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5755..5775
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(5894..6081,6082..6093)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     rep_origin      complement(6154..6742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6234..6253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6913..7704,7705..7773))
                     /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     7536..7555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7774..7878)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7957..7976)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    join(8168..8179,8180..8317)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     enhancer        8326..8705
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8706..8903,1..6)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8860..8880
                     /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 cgccgacaat ggcgatatgg tgagagtgga cgtgttctgt aaggtggata agaagggcaa
     3661 gaatcagtac tttatcgtgc ctatctatgc ctggcaggtg gccgagaaca tcctgccaga
     3721 catcgattgc aagggctaca gaatcgacga tagctataca ttctgttttt ccctgcacaa
     3781 gtatgacctg atcgccttcc agaaggatga gaagtccaag gtggagtttg cctactatat
     3841 caattgcgac tcctctaacg gcaggttcta cctggcctgg cacgataagg gcagcaagga
     3901 gcagcagttt cgcatctcca cccagaatct ggtgctgatc cagaagtatc aggtgaacga
     3961 gctgggcaag gagatcaggc catgtcggct gaagaagcgc ccacccgtgc gggaggataa
     4021 aagaaccgcc gacggcagcg aattcgagcc caagaagaag aggaaagtct aaccggtcat
     4081 catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     4141 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4201 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4261 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     4321 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg
     4381 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4441 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4501 tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4561 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4621 ggtttgcgta ttgggcgcta cagacatgat aagatacatt gatgagtttg gacaaaccac
     4681 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4741 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat
     4801 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
     4861 catagaaggc ggcggtggaa tcgaaatctc gtagcacgtg tcagtcctgc tcctcggcca
     4921 cgaagtgctt agccctccca cacataacca gagggcagca attcacgaat cccaactgcc
     4981 gtcggctgtc catcactgtc cttcactatg gctttgatcc caggatgcag atcgagaagc
     5041 acctgtcggc accgtccgca ggggctcaag atgcccctgt tctcatttcc gatcgcgacg
     5101 atacaagtca ggttgccagc tgccgcagca gcagcagtgc ccagcaccac gagttctgca
     5161 caaggtcccc cagtaaaatg atatacattg acaccagtga agatgcggcc gtcgctagag
     5221 agagctgcgc tggcgacgct gtagtcttca gagatgggga tgctgttgat tgtagccgtt
     5281 gctctttcaa tgagggtgga ttcttcttga gacaaaggct tggccatggt ttagttcctc
     5341 accttgtcgt attatactat gccgatatac tatgccgatg attaattgtc aacacgtgct
     5401 gatcagatcc gaaaatggat atacaagctc ccgggagctt tttgcaaaag cctaggcctc
     5461 caaaaaagcc tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg
     5521 cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta
     5581 ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc
     5641 tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat
     5701 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta
     5761 actgacacac attccacaga attcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5821 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5881 ggaaagaaca tgtaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt
     5941 aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg
     6001 atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat
     6061 caagtaaaat tactcaagta ctttacacct ctgcatgtga gcaaaaggcc agcaaaaggc
     6121 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6181 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6241 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6301 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6361 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6421 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6481 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6541 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6601 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6661 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6721 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacactca
     6781 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6841 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6901 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6961 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7021 accatctggc cccagtgctg caatgatacc gcgggaccca cgctcaccgg ctccagattt
     7081 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7141 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7201 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7261 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7321 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7381 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7441 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7501 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7561 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7621 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7681 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7741 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7801 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7861 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     7921 agatcgatct cccgatcccc tagggtcgac tctcagtaca atctgctctg atgccgcata
     7981 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     8041 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     8101 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgggtac cggcatatgg
     8161 ttcttgacag aggtgtaaaa agtactcaaa aattttactc aagtgaaagt acaagtactt
     8221 agggaaaatt ttactcaatt aaaagtaaaa gtatctggct agaatcttac ttgagtaaaa
     8281 gtaaaaaagt actccattaa aattgtactt gagtattaac gcgttgacat tgattattga
     8341 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     8401 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     8461 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     8521 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     8581 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     8641 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     8701 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     8761 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     8821 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     8881 tacggtggga ggtctatata agc
//
