LOCUS       sequence-454006-        8837 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses domain inlaid Nme2 nCas9 (D16A)-i8-ABE8e base editor with 
            linker-10 and E932R mutation in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8837)
  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 8837)
  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: pEJS2706-CMV-Nme2-ABE8e-i8-L10-E932R
FEATURES             Location/Qualifiers
     source          1..8837
                     /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             3983..4003
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4012..4029
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4052..4069)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4058..4282
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4353..4369)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4353..4369)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4366..4388)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4377..4393
                     /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(4401..4407,4408..4425,4426..4431))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4446..4467
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    4584..4705
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4595..4614)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4649..4668
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4863..5261)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(5280..5327)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5375..5704)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5389..5524
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5443..5462)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5689..5709
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(5828..6015,6016..6027)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     rep_origin      complement(6088..6676)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6168..6187)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6847..7638,7639..7707))
                     /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     7470..7489
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7708..7812)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7891..7910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    join(8102..8113,8114..8251)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     enhancer        8260..8639
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8640..8837,1..6)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8794..8814
                     /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 gggactggcc atcggaatcg catccgtggg atgggctatg gtggagatcg acgaggagga
      241 gaatcctatc cggctgatcg atctgggcgt gagagtgttt gagagggccg aggtgccaaa
      301 gaccggcgat 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 caagccttct ggcggttcag gtggatcagg cggtagctct gaggtggagt tttcccacga
     2581 gtactggatg agacatgccc tgaccctggc caagagggca cgcgatgaga gggaggtgcc
     2641 tgtgggagcc gtgctggtgc tgaacaatag agtgatcggc gagggctgga acagagccat
     2701 cggcctgcac gacccaacag cccatgccga aattatggcc ctgagacagg gcggcctggt
     2761 catgcagaac tacagactga ttgacgccac cctgtacgtg acattcgagc cttgcgtgat
     2821 gtgcgccggc gccatgatcc actctaggat cggccgcgtg gtgtttggcg tgaggaacag
     2881 caaacggggc gccgcaggct ccctgatgaa cgtgctgaac taccccggca tgaatcaccg
     2941 cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc gccctgctgt gcgacttcta
     3001 ccggatgcct agacaggtgt tcaatgctca gaagaaggcc cagagctcca tcaacgagac
     3061 acctggcaca agcgagagcg caacagagtt tgaggaggcc gataccccag agaagctgag
     3121 gacactgctg gcagagaagc tgtctagcag gccagaggca gtgcacgagt acgtgacccc
     3181 actgttcgtg tccagggcac ccaatcggaa gatgtctggc gcccacaagg acacactgag
     3241 aagcgccaag aggtttgtga agcacaacga gaagatctcc gtgaagagag tgtggctgac
     3301 cgagatcaag ctggccgatc tggagaacat ggtgaattac aagaacggca gggagatcga
     3361 gctgtatgag gccctgaagg caaggctgga ggcctacgga ggaaatgcca agcaggcctt
     3421 cgacccaaag gataacccct tttataagaa gggaggacag ctggtgaagg ccgtgcgggt
     3481 ggagaagacc cagcgtagcg gcgtgctgct gaataagaag aacgcctaca caatcgccga
     3541 caatggcgat atggtgagag tggacgtgtt ctgtaaggtg gataagaagg gcaagaatca
     3601 gtactttatc gtgcctatct atgcctggca ggtggccgag aacatcctgc cagacatcga
     3661 ttgcaagggc tacagaatcg acgatagcta tacattctgt ttttccctgc acaagtatga
     3721 cctgatcgcc ttccagaagg atgagaagtc caaggtggag tttgcctact atatcaattg
     3781 cgactcctct aacggcaggt tctacctggc ctggcacgat aagggcagca aggagcagca
     3841 gtttcgcatc tccacccaga atctggtgct gatccagaag tatcaggtga acgagctggg
     3901 caaggagatc aggccatgtc ggctgaagaa gcgcccaccc gtgcgggagg ataaaagaac
     3961 cgccgacggc agcgaattcg agcccaagaa gaagaggaaa gtctaaccgg tcatcatcac
     4021 catcaccatt gagtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     4081 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4141 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4201 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4261 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctcgataccg
     4321 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4381 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctagggt
     4441 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4501 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4561 cgtattgggc gctacagaca tgataagata cattgatgag tttggacaaa ccacaactag
     4621 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     4681 cattataagc tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc
     4741 gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga
     4801 aggcggcggt ggaatcgaaa tctcgtagca cgtgtcagtc ctgctcctcg gccacgaagt
     4861 gcttagccct cccacacata accagagggc agcaattcac gaatcccaac tgccgtcggc
     4921 tgtccatcac tgtccttcac tatggctttg atcccaggat gcagatcgag aagcacctgt
     4981 cggcaccgtc cgcaggggct caagatgccc ctgttctcat ttccgatcgc gacgatacaa
     5041 gtcaggttgc cagctgccgc agcagcagca gtgcccagca ccacgagttc tgcacaaggt
     5101 cccccagtaa aatgatatac attgacacca gtgaagatgc ggccgtcgct agagagagct
     5161 gcgctggcga cgctgtagtc ttcagagatg gggatgctgt tgattgtagc cgttgctctt
     5221 tcaatgaggg tggattcttc ttgagacaaa ggcttggcca tggtttagtt cctcaccttg
     5281 tcgtattata ctatgccgat atactatgcc gatgattaat tgtcaacacg tgctgatcag
     5341 atccgaaaat ggatatacaa gctcccggga gctttttgca aaagcctagg cctccaaaaa
     5401 agcctcctca ctacttctgg aatagctcag aggcagaggc ggcctcggcc tctgcataaa
     5461 taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg
     5521 ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca
     5581 tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac taattgagat
     5641 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac
     5701 acacattcca cagaattcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5761 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5821 aacatgtaag tgatctccaa aaaataagta ctttttgact gtaaataaaa ttgtaaggag
     5881 taaaaagtac ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt attgattttt
     5941 aattgtactc aagtaaagta aaaatcccca aaaataatac ttaagtacag taatcaagta
     6001 aaattactca agtactttac acctctgcat gtgagcaaaa ggccagcaaa aggccaggaa
     6061 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     6121 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6181 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6241 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6301 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6361 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6421 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6481 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6541 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6601 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6661 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgaca ctcagtggaa
     6721 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6781 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6841 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6901 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6961 tggccccagt gctgcaatga taccgcggga cccacgctca ccggctccag atttatcagc
     7021 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     7081 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7141 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7201 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7261 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7321 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7381 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7441 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7501 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7561 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7621 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7681 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7741 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7801 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcg
     7861 atctcccgat cccctagggt cgactctcag tacaatctgc tctgatgccg catagttaag
     7921 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     7981 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     8041 ttttgcgctg cttcgcgatg tacgggccag atatacgcgg gtaccggcat atggttcttg
     8101 acagaggtgt aaaaagtact caaaaatttt actcaagtga aagtacaagt acttagggaa
     8161 aattttactc aattaaaagt aaaagtatct ggctagaatc ttacttgagt aaaagtaaaa
     8221 aagtactcca ttaaaattgt acttgagtat taacgcgttg acattgatta ttgactagtt
     8281 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     8341 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     8401 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     8461 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     8521 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     8581 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     8641 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     8701 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     8761 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     8821 gggaggtcta tataagc
//
