LOCUS       sequence-453997-        8840 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses domain inlaid eNme2-C nCas9 (D16A)-i1-ABE8e base editor 
            with linker-10 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8840)
  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 8840)
  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: pEJS2712-CMV-eNme2-C-ABE8e-i1-L10
FEATURES             Location/Qualifiers
     source          1..8840
                     /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             3986..4006
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4015..4032
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4055..4072)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4061..4285
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4356..4372)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4356..4372)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4369..4391)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4380..4396
                     /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(4404..4410,4411..4428,4429..4434))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4449..4470
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    4587..4708
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4598..4617)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4652..4671
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4866..5264)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(5283..5330)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5378..5707)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5392..5527
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5446..5465)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5692..5712
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(5831..6018,6019..6030)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     rep_origin      complement(6091..6679)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6171..6190)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6850..7641,7642..7710))
                     /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     7473..7492
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7711..7815)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7894..7913)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    join(8105..8116,8117..8254)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     enhancer        8263..8642
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8643..8840,1..6)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8797..8817
                     /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 atgcagcatt caagtcaaac ccaatcaatt acatcctggg
      181 actggcaatc ggaatcgcat ccgtgggatg ggctatggtg gagatcgacg aggaggggaa
      241 tcctatccgg ctgatcgatc tgggcgtgag agtgtttgag agggccgagg tgccaaagac
      301 cggcgattct ctggctatgg cccggagact ggcacggagc gtgaggcgcc tgacacggag
      361 aagggcacac aggctgctga gggcacgccg gctgctgaag agagagggcg tgctgcaggc
      421 agcagacttc gatgagaatg gcctgatcac gagcttgcca aacaccccct ggcagctgag
      481 agcagccgcc ctggacagga agctgacacc actggagtgg tctgccgtgc tgctgcacct
      541 gatcaagcac cgcggctacc tgagccagcg gaagaacgag ggagagacag cagccaagga
      601 gctgggcgcc ctgctgaagg gagtggccaa caatgcccac gccctgcaga ccggcgattt
      661 caggacacct gccgagctgg ccctgaataa gtttgagaag gagtccggcc acatcagaaa
      721 ccagaggggc gactatagcc acaccttctc ccgcaaggat ctgcaggccg agctgatcct
      781 gctgttcgag aagcagaagg agtttggcaa tccacacgtg agcggaggcc tgaaggaggg
      841 aatcgagacc ctgctgatga cacagaggcc tgccctgtcc ggcgacgcag tgcagaagat
      901 gctggggcac tgcaccctcg agcctacaga gccaaaggcc gccaagaaca cctacacagc
      961 cgagcggttt atctggctga caaagctgaa caatctgaga atcctggagc agtctggcgg
     1021 ttcaggtgga tcaggcggta gctctgaggt ggagttttcc cacgagtact ggatgagaca
     1081 tgccctgacc ctggccaaga gggcacgcga tgagagggag gtgcctgtgg gagccgtgct
     1141 ggtgctgaac aatagagtga tcggcgaggg ctggaacaga gccatcggcc tgcacgaccc
     1201 aacagcccat gccgaaatta tggccctgag acagggcggc ctggtcatgc agaactacag
     1261 actgattgac gccaccctgt acgtgacatt cgagccttgc gtgatgtgcg ccggcgccat
     1321 gatccactct aggatcggcc gcgtggtgtt tggcgtgagg aacagcaaac ggggcgccgc
     1381 aggctccctg atgaacgtgc tgaactaccc cggcatgaat caccgcgtcg aaattaccga
     1441 gggaatcctg gcagatgaat gtgccgccct gctgtgcgac ttctaccgga tgcctagaca
     1501 ggtgttcaat gctcagaaga aggcccagag ctccatcaac gagacacctg gcacaagcga
     1561 gagcgcaaca ggatccgaga ggccactgac cgacacagag aggtccaccc tgatggatga
     1621 gccttaccgg aagtctaaac tgacatatgc ccaggccaga aagctgctgg gcctggagga
     1681 caccgccttc tttaagggcc tgagatacgg caaggataat gccgaggcct ccacactgat
     1741 ggagatgaag gcctatcacg ccatctctcg cgccctggag aaggagggcc tgaaggacaa
     1801 gaagtccccc ctgaacctga gctccgagct gcaggatgag atcggcaccg ccttctctct
     1861 gtttaagacc gacgaggata tcacaggccg cctgaaggac agggtgcagc ctgagatcct
     1921 ggaggccctg ctgaagcaca tctctttcga taagtttgtg cagatcagcc tgaaggccct
     1981 gagaaggatc gtgccactga tggagcaggg caagcggtac gacgaggcct gcgccgagat
     2041 ctacggcgtt cactatggca agaagaacac agaggagaag atctatctgc cccctatccc
     2101 tgccgacgag atcagaaatc ctgtggtgct gagggccctg tcccaggcaa gaaaagtgat
     2161 caacggagtg gtgcgccggt acggatctcc agcccggatc cacatcgaga ccgccagaga
     2221 agtgggcaag agcttcaagg accggaagga gatcgcgaag agacaggagg agaatcgcaa
     2281 ggatcgggag aaggccgccg ccaagtttag ggagtacttc cctaactttg tgggcgagcc
     2341 aaagtctaag gacatcctga agctgcgcct gtacgagcag cagcacggca agtgtctgta
     2401 tagcggcaaa gagatcaatc tggtgcggct gaacgagaag ggctatgtgg agatcgatca
     2461 cgccctgcct ttctccagaa cctgggacga ttcttttaac aataaggtgc tggtgctggg
     2521 cagcgagaac cagaataagg gcaatcagac accatacgag tatttcaatg gcaaggacaa
     2581 ctccagggag tggcaggagt tcaaggcccg cgtggagacc tctagatttc ccagtagcaa
     2641 gaagcagcgg atcctgctgc agaagttcga cgaggatggc tttaaggagt gcaacctgaa
     2701 tgacaccaga tacgtgaacc ggttcctgtg ccagtttgtg gccgatcaca tcctgctgac
     2761 cggcaagggc aagagaaggg tggtcgcctc taatggccag atcacaaacc tgctgagggg
     2821 gttttggaga ctgaggaagg tgcgggcaga gaatgacaga caccacgcac tggatgcagt
     2881 ggtggtggca tgcagcaccg tggcaatgca gcagaagatc acaagattcg tgaggtataa
     2941 ggagatgaac gcctttgacg gcaagaccgt cgataaggag acaggcaagg tgctgtacca
     3001 gaagacccac ttcccccagc cttgggagtt ctttgcccag gaagttatga tccgggtgtt
     3061 cggcaagcca gacggcaagc ctgagtttga ggaggccgat accccagaga agctgaggac
     3121 actgctggca gagaagctgt ctagcaggcc agaggcagtg cacgagtacg tgaccccgct
     3181 gttcgtgtcc agggcaccca atcggaagat gtctggcgcc cacaaggaca cactgagaag
     3241 cgccaagagg tttgtgaagc acaacgagaa gatctccgtg aagagagtgt ggctgaccga
     3301 gatcaagctg gccgatctgg agaacatggt gaattacaag aacggcaggg agatcgagct
     3361 gtatgaggcc ctgaaggcaa ggctggaggc ctacggagga aatgccaagc aggccttcga
     3421 cccaaaggat aacccctttt ataagaaggg aggacagctg gtgaaggccg tgcgggtgga
     3481 gaagacccag aagagcggcg tgctgctgaa taagaagaac gcctacacaa tcgccgacaa
     3541 tggtgatatg gtgagagtgg acgtgttctg taaggtggat aagaagggca agaatcagta
     3601 ctttatcgtg cctatctatg cctggcaggt ggccgagaac atcctgccag acatcgattg
     3661 caagggctac agaatcgacg atagctatac attctgtttt tccctgcaca agtatgacct
     3721 gatcgccttc cagaaggatg agaagtccaa ggtggagttt gcctactata tcaattgcga
     3781 ctcctctagc ggcgggttct acctggcctg gcacgataag ggcagcaggg agcagcggtt
     3841 tcgcatctcc acccagaatc tggcgctgat ccagaagtat caggtgaacg agctgggcaa
     3901 ggagatcagg ccatgtcggc tgaagaagcg cccacccgtg cggtctggcg gctcaaaaag
     3961 aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg aaagtctaac cggtcatcat
     4021 caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     4081 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4141 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4201 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     4261 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata
     4321 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4381 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag
     4441 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4501 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4561 ttgcgtattg ggcgctacag acatgataag atacattgat gagtttggac aaaccacaac
     4621 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     4681 aaccattata agctgcaata aacaagttgg ggtgggcgaa gaactccagc atgagatccc
     4741 cgcgctggag gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat
     4801 agaaggcggc ggtggaatcg aaatctcgta gcacgtgtca gtcctgctcc tcggccacga
     4861 agtgcttagc cctcccacac ataaccagag ggcagcaatt cacgaatccc aactgccgtc
     4921 ggctgtccat cactgtcctt cactatggct ttgatcccag gatgcagatc gagaagcacc
     4981 tgtcggcacc gtccgcaggg gctcaagatg cccctgttct catttccgat cgcgacgata
     5041 caagtcaggt tgccagctgc cgcagcagca gcagtgccca gcaccacgag ttctgcacaa
     5101 ggtcccccag taaaatgata tacattgaca ccagtgaaga tgcggccgtc gctagagaga
     5161 gctgcgctgg cgacgctgta gtcttcagag atggggatgc tgttgattgt agccgttgct
     5221 ctttcaatga gggtggattc ttcttgagac aaaggcttgg ccatggttta gttcctcacc
     5281 ttgtcgtatt atactatgcc gatatactat gccgatgatt aattgtcaac acgtgctgat
     5341 cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct aggcctccaa
     5401 aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg gcctctgcat
     5461 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg
     5521 gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt
     5581 gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga
     5641 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact
     5701 gacacacatt ccacagaatt cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5761 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5821 aagaacatgt aagtgatctc caaaaaataa gtactttttg actgtaaata aaattgtaag
     5881 gagtaaaaag tacttttttt tctaaaaaaa tgtaattaag taaaagtaaa agtattgatt
     5941 tttaattgta ctcaagtaaa gtaaaaatcc ccaaaaataa tacttaagta cagtaatcaa
     6001 gtaaaattac tcaagtactt tacacctctg catgtgagca aaaggccagc aaaaggccag
     6061 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6121 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6181 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6241 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6301 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6361 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6421 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6481 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6541 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6601 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6661 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acactcagtg
     6721 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6781 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6841 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6901 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6961 atctggcccc agtgctgcaa tgataccgcg ggacccacgc tcaccggctc cagatttatc
     7021 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7081 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7141 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7201 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7261 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7321 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7381 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7441 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7501 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7561 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7621 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7681 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7741 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7801 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     7861 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
     7921 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     7981 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     8041 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgggtaccgg catatggttc
     8101 ttgacagagg tgtaaaaagt actcaaaaat tttactcaag tgaaagtaca agtacttagg
     8161 gaaaatttta ctcaattaaa agtaaaagta tctggctaga atcttacttg agtaaaagta
     8221 aaaaagtact ccattaaaat tgtacttgag tattaacgcg ttgacattga ttattgacta
     8281 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     8341 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     8401 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     8461 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     8521 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     8581 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     8641 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     8701 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     8761 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     8821 ggtgggaggt ctatataagc
//
