LOCUS       sequence-454000-        8861 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses domain inlaid Nme2Smu nCas9 (D16A)-i1-ABE8e base editor 
            with linker-10 and D56R mutation in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8861)
  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 8861)
  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: pEJS2709-CMV-Nme2Smu-ABE8e-i1-L10-D56R
FEATURES             Location/Qualifiers
     source          1..8861
                     /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      3545..3554
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4007..4027
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4036..4053
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4076..4093)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4082..4306
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4377..4393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4377..4393)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4390..4412)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4401..4417
                     /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(4425..4431,4432..4449,4450..4455))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4470..4491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    4608..4729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4619..4638)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4673..4692
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4887..5285)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(5304..5351)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5399..5728)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5413..5548
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5467..5486)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5713..5733
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(5852..6039,6040..6051)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     rep_origin      complement(6112..6700)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6192..6211)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6871..7662,7663..7731))
                     /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     7494..7513
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7732..7836)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7915..7934)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    join(8126..8137,8138..8275)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     enhancer        8284..8663
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8664..8861,1..6)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8818..8838
                     /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 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 agcagtctgg
     1021 cggttcaggt ggatcaggcg gtagctctga ggtggagttt tcccacgagt actggatgag
     1081 acatgccctg accctggcca agagggcacg cgatgagagg gaggtgcctg tgggagccgt
     1141 gctggtgctg aacaatagag tgatcggcga gggctggaac agagccatcg gcctgcacga
     1201 cccaacagcc catgccgaaa ttatggccct gagacagggc ggcctggtca tgcagaacta
     1261 cagactgatt gacgccaccc tgtacgtgac attcgagcct tgcgtgatgt gcgccggcgc
     1321 catgatccac tctaggatcg gccgcgtggt gtttggcgtg aggaacagca aacggggcgc
     1381 cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg aatcaccgcg tcgaaattac
     1441 cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc gacttctacc ggatgcctag
     1501 acaggtgttc aatgctcaga agaaggccca gagctccatc aacgagacac ctggcacaag
     1561 cgagagcgca acaggatccg agaggccact gaccgacaca gagagggcca ccctgatgga
     1621 tgagccttac cggaagtcta agctgacata tgcccaggcc agaaagctgc tgggcctgga
     1681 ggacaccgcc ttctttaagg gcctgagata cggcaaggat aatgccgagg cctccacact
     1741 gatggagatg aaggcctatc acgccatctc tcgcgccctg gagaaggagg gcctgaagga
     1801 caagaagtcc cccctgaacc tgagctccga gctgcaggat gagatcggca ccgccttctc
     1861 tctgtttaag accgacgagg atatcacagg ccgcctgaag gacagggtgc agcctgagat
     1921 cctggaggcc ctgctgaagc acatctcttt cgataagttt gtgcagatca gcctgaaggc
     1981 cctgagaagg atcgtgccac tgatggagca gggcaagcgg tacgacgagg cctgcgccga
     2041 gatctacggc gatcactatg gcaagaagaa cacagaggag aagatctatc tgccccctat
     2101 ccctgccgac gagatcagaa atcctgtggt gctgagggcc ctgtcccagg caagaaaagt
     2161 gatcaacgga gtggtgcgcc ggtacggatc tccagcccgg atccacatcg agaccgccag
     2221 agaagtgggc aagagcttca aggaccggaa ggagatcgag aagagacagg aggagaatcg
     2281 caaggatcgg gagaaggccg ccgccaagtt tagggagtac ttccctaact ttgtgggcga
     2341 gccaaagtct aaggacatcc tgaagctgcg cctgtacgag cagcagcacg gcaagtgtct
     2401 gtatagcggc aaggagatca atctggtgcg gctgaacgag aagggctatg tggagatcga
     2461 tcacgccctg cctttctcca gaacctggga cgattctttt aacaataagg tgctggtgct
     2521 gggcagcgag aaccagaata agggcaatca gacaccatac gagtatttca atggcaagga
     2581 caactccagg gagtggcagg agttcaaggc ccgcgtggag acctctagat ttcccaggag
     2641 caagaagcag cggatcctgc tgcagaagtt cgacgaggat ggctttaagg agtgcaacct
     2701 gaatgacacc agatacgtga accggttcct gtgccagttt gtggccgatc acatcctgct
     2761 gaccggcaag ggcaagagaa gggtgttcgc ctctaatggc cagatcacaa acctgctgag
     2821 gggattttgg ggactgagga aggtgcgggc agagaatgac agacaccacg cactggatgc
     2881 agtggtggtg gcatgcagca ccgtggcaat gcagcagaag atcacaagat tcgtgaggta
     2941 taaggagatg aacgcctttg acggcaagac catcgataag gagacaggca aggtgctgca
     3001 ccagaagacc cacttccccc agccttggga gttctttgcc caggaagtga tgatccgggt
     3061 gttcggcaag ccagacggca agcctgagtt 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 caggagagcg gcgtgctgct gaataagaag aacgcctaca caatcgccga
     3541 caacgccacc atggtgcggg tggacgtgta caccaaggcc ggcaagaact acctggttcc
     3601 tgtgtacgtg tggcaggtgg cccagggcat cttacccaac cgcgccgtga ccagcggcaa
     3661 gtccgaggct gactgggacc tgatcgatga gagcttcgag ttcaagttct ctctgtcccg
     3721 gggagatctc gtggaaatga tctccaacaa gggcagaatc ttcggctact acaacggcct
     3781 ggacagagcc aacggctcta ttggaattag agagcacgac ctagagaaga gcaagggcaa
     3841 agacggcgtg catagagtgg gagtgaaaac agctacagca tttaacaagt accacgtgga
     3901 tcccctgggc aaagagatcc acagatgcag cagcgaaccc agacctacac tgaaaatcaa
     3961 gtctaagaag gaggataaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag
     4021 gaaagtctaa ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg
     4081 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     4141 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     4201 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     4261 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     4321 agaaccagct ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4381 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4441 agcataaagt gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4561 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctaca gacatgataa gatacattga
     4621 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     4681 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga
     4741 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     4801 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt agcacgtgtc
     4861 agtcctgctc ctcggccacg aagtgcttag ccctcccaca cataaccaga gggcagcaat
     4921 tcacgaatcc caactgccgt cggctgtcca tcactgtcct tcactatggc tttgatccca
     4981 ggatgcagat cgagaagcac ctgtcggcac cgtccgcagg ggctcaagat gcccctgttc
     5041 tcatttccga tcgcgacgat acaagtcagg ttgccagctg ccgcagcagc agcagtgccc
     5101 agcaccacga gttctgcaca aggtccccca gtaaaatgat atacattgac accagtgaag
     5161 atgcggccgt cgctagagag agctgcgctg gcgacgctgt agtcttcaga gatggggatg
     5221 ctgttgattg tagccgttgc tctttcaatg agggtggatt cttcttgaga caaaggcttg
     5281 gccatggttt agttcctcac cttgtcgtat tatactatgc cgatatacta tgccgatgat
     5341 taattgtcaa cacgtgctga tcagatccga aaatggatat acaagctccc gggagctttt
     5401 tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggcag
     5461 aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
     5521 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
     5581 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     5641 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     5701 gggactttcc acaccctaac tgacacacat tccacagaat tcactgactc gctgcgctcg
     5761 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5821 gaatcagggg ataacgcagg aaagaacatg taagtgatct ccaaaaaata agtacttttt
     5881 gactgtaaat aaaattgtaa ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa
     5941 gtaaaagtaa aagtattgat ttttaattgt actcaagtaa agtaaaaatc cccaaaaata
     6001 atacttaagt acagtaatca agtaaaatta ctcaagtact ttacacctct gcatgtgagc
     6061 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     6121 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6181 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6241 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6301 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6361 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6421 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6481 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6541 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6601 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6661 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6721 tacggggtct gacactcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6781 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6841 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6901 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6961 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gggacccacg
     7021 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     7081 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     7141 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     7201 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     7261 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     7321 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     7381 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     7441 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     7501 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7561 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7621 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7681 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7741 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7801 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7861 tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc tcagtacaat
     7921 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     7981 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     8041 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     8101 gcgggtaccg gcatatggtt cttgacagag gtgtaaaaag tactcaaaaa ttttactcaa
     8161 gtgaaagtac aagtacttag ggaaaatttt actcaattaa aagtaaaagt atctggctag
     8221 aatcttactt gagtaaaagt aaaaaagtac tccattaaaa ttgtacttga gtattaacgc
     8281 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     8341 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     8401 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     8461 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     8521 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     8581 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     8641 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     8701 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     8761 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     8821 aaatgggcgg taggcgtgta cggtgggagg tctatataag c
//
