LOCUS       sequence-351817-        7106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression  of base editor in mammalian cells .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7106)
  AUTHORS   Mok BY, Kotrys AV, Raguram A, Huang TP, Mootha VK, Liu DR
  TITLE     CRISPR-free base editors with enhanced activity and expanded 
            targeting scope in mitochondrial and nuclear DNA.
  JOURNAL   Nat Biotechnol. 2022 Sep;40(9):1378-1387. doi: 
            10.1038/s41587-022-01256-8. Epub 2022 Apr 4.
  PUBMED    35379961
REFERENCE   2  (bases 1 to 7106)
  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"; doi: 
            "10.1038/s41587-022-01256-8"; journalName: "Nat Biotechnol"; date: 
            "2022-09"; volume: "40"; issue: "9"; pages: "1378-1387"
COMMENT     Sequence Label: ND4.2-Right DdCBE-G1397-C-wildtype
FEATURES             Location/Qualifiers
     source          1..7106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             904..981
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MASVLTPLLLRGLTGSARRLPVPRAK"
     CDS             994..1059
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2890..3141
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      3338..3766
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3425..3444)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3635..3656
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3775..3795)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3780..4109
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3960..4095
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4022..4041
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4157..4204
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4223..4621
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4779..4900
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4816..4835)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4870..4889
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4949..4965)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4949..4965)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4962..4984)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4973..4989
                     /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(4997..5003,5004..5021,5022..5027))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5042..5063
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5180..5197)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5351..5939)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5431..5450)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6110..6901,6902..6970))
                     /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     6733..6752
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6971..7075)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 accatggcct ccgttctgac cccgctgctg ctgcgtggcc tgaccggctc cgctcgtcgt
      961 ctgccagttc cgcgtgcgaa aatccattcc ctggactaca aagaccatga cggtgattat
     1021 aaagatcatg acatcgatta caaggatgac gatgacaaga tggacatcgc ggatctgcgt
     1081 accctgggtt acagccagca gcagcaggag aagatcaagc cgaaggtgcg cagcaccgtg
     1141 gctcagcacc acgaagccct ggtgggccac ggtttcaccc acgctcacat cgtggccctg
     1201 agccagcacc cagccgcgct gggcaccgtg gccgtgaaat atcaggacat gattgctgcc
     1261 ctgccagagg ccacccatga agctattgtg ggcgtgggca agcagtggag cggtgctcgt
     1321 gcgctggagg cgctgctgac cgtggctggt gaactgcgtg gtccgccgct gcagctggat
     1381 accggtcagc tgctgaaaat tgccaaacgt ggcggtgtga ccgcggtgga agccgtgcat
     1441 gcttggcgta atgctctgac cggtgcgccg ctgaacctca cacccgaaca ggttgttgct
     1501 atcgccagca acaacggagg aaagcaggcc cttgagacag tgcaggccct gctccccgtg
     1561 ctgtgccagg cccacggact gacccctcaa caggtggttg ctatcgcctc caacaacggc
     1621 ggcaagcagg ccctggaaac cgtgcagagg ctgctgcccg tgctgtgtca ggcccacggc
     1681 ctgacccctc agcaggtcgt ggccatcgcc agcaacaacg gcggaaagca ggcacttgaa
     1741 accgtccaaa gactgctgcc cgtgctgtgc caggcccatg gactgacacc cgagcaggtg
     1801 gtggccattg ccagccacga cggcggaaag caagctctgg agaccgttca agctctgctg
     1861 ccagtgctgt gccaggccca cggactgaca ccagagcagg tggtggctat cgcctctaac
     1921 aacggcggca agcaggccct tgaaaccgtg caggctctgc tgcctgtgct gtgccaggct
     1981 cacggcctga cacctgagca ggtcgtggcc atcgcctcta atatcggcgg caagcaggcc
     2041 ctggagacag tgcaggctct gctgcctgtt ctgtgtcagg cccacggcct gacccctgag
     2101 caggtggtgg ccatcgccag caacggcggc ggcaagcagg ccctcgagac agtgcagaga
     2161 ctgctgcccg tgctgtgcca ggcccacggc ctgacacctg agcaggtggt ggctatcgcc
     2221 agcaacggcg gcggcaaaca agccctggaa acagtgcaag ctcttctgcc tgtgctgtgt
     2281 caggcccacg gactgacccc tcagcaggtg gtggccatcg ccagcaacat cggcggtaag
     2341 caggccctgg aaaccgtgca gcggctgctg cctgtgctgt gccaggccca cggcctgacc
     2401 cctgagcaag tggtggccat cgctagcaat ggaggcggca aacaggccct ggaaaccgtg
     2461 cagcggctgc tgcctgtgct gtgccaggcc cacggcctga ccccacaaca agtggtggcc
     2521 atcgccagca acaatggcgg caaacaggcc ctggaaaccg tgcagagact gctgcctgtg
     2581 ctgtgccagg cccacggcct gaccccccag caggtggtgg ccatcgcctc caatatcggc
     2641 gggagacccg ccctggaaag cattgtggct cagctgagcc gtccagaccc ggcgctggcg
     2701 gctctgacca acgatcacct ggtggcgctg gcttgcctgg gcggtcgtcc ggccctggat
     2761 gccgtgaaga aaggcctggg tggatccgcc attccagtga agcgcggcgc taccggtgaa
     2821 accaaagtgt ttaccggtaa cagcaacagc ccgaagagcc cgaccaaagg cggttgctct
     2881 ggcggctcaa ctaatctgag cgacatcatt gagaaggaga ctgggaaaca gctggtcatt
     2941 caggagtcca tcctgatgct gcctgaggag gtggaggaag tgatcggcaa caagccagag
     3001 tctgacatcc tggtgcacac cgcctacgac gagtccacag atgagaatgt gatgctgctg
     3061 acctctgacg cccccgagta taagccttgg gccctggtca tccaggattc taacggcgag
     3121 aataagatca agatgctgtg ataaggggtc tttgtcctct gtactgtctc tctccttgcc
     3181 cctaacccaa aaagcttcat ttttctgtgt aggctgcaca agagccttga ttgaagatat
     3241 attctttctg aacagtattt aaggtttcca ataaaatgta cacccctcag aacttctgag
     3301 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     3361 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3421 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3481 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3541 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3601 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3661 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3721 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     3781 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3841 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3901 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3961 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4021 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     4081 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     4141 gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata
     4201 cgacaaggtg aggaactaaa ccatggccaa gcctttgtct caagaagaat ccaccctcat
     4261 tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag
     4321 cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg
     4381 gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct
     4441 gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg
     4501 ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg
     4561 acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta
     4621 agcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat tccaccgccg
     4681 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4741 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4801 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4861 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4921 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4981 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5041 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5101 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5161 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5221 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5281 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5341 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5401 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5461 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5521 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5581 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5641 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5701 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5761 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5821 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5881 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5941 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6001 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6061 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6121 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6181 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6241 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6301 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6361 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6421 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6481 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6541 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6601 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6661 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6721 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6781 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6841 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6901 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6961 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7021 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7081 ttccccgaaa agtgccacct gacgtc
//
