LOCUS       sequence-351812-        8012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression  of base editor in mammalian cells and for cell sorting .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8012)
  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 8012)
  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-Left TALE-G1397-N-dead-mCherry
FEATURES             Location/Qualifiers
     source          1..8012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(164..183)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        355..734
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        735..938
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     889..909
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     983..1002
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        983..1001
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1024..1101
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MASVLTPLLLRGLTGSARRLPVPRAK"
     CDS             1114..1179
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3142..3390
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3400..3456
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     rep_origin      4364..4792
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4451..4470)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4661..4682
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4801..4821)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4806..5135
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4986..5121
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5048..5067
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5183..5230
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5249..5647
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5805..5926
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5842..5861)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5896..5915
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5975..5991)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5975..5991)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5988..6010)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5999..6015
                     /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(6023..6029,6030..6047,6048..6053))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6068..6089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6206..6223)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6377..6965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6457..6476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7136..7927,7928..7996))
                     /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     7759..7778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7997..8012,1..89))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
       61 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
      121 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
      181 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      241 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      301 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      361 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      421 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      481 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      541 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      601 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      661 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      721 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      781 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      841 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      901 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      961 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
     1021 accatggcct ccgttctgac cccgctgctg ctgcgtggcc tgaccggctc cgctcgtcgt
     1081 ctgccagttc cgcgtgcgaa aatccattcc ctggactaca aagaccatga cggtgattat
     1141 aaagatcatg acatcgatta caaggatgac gatgacaaga tggacatcgc ggatctgcgt
     1201 accctgggtt acagccagca gcagcaggag aagatcaagc cgaaggtgcg cagcaccgtg
     1261 gctcagcacc acgaagccct ggtgggccac ggtttcaccc acgctcacat cgtggccctg
     1321 agccagcacc cagccgcgct gggcaccgtg gccgtgaaat atcaggacat gattgctgcc
     1381 ctgccagagg ccacccatga agctattgtg ggcgtgggca agcagtggag cggtgctcgt
     1441 gcgctggagg cgctgctgac cgtggctggt gaactgcgtg gtccgccgct gcagctggat
     1501 accggtcagc tgctgaaaat tgccaaacgt ggcggtgtga ccgcggtgga agccgtgcat
     1561 gcttggcgta atgctctgac cggtgcgccg ctgaacttaa cgccggaaca agttgttgca
     1621 attgccagca acaacggcgg caagcaagct ctggaaaccg tgcaggccct gcttccagtc
     1681 ctgtgtcagg cccacggcct gacccctcag caggtggtcg ccatcgcctc taacatcggc
     1741 ggaaagcagg ctctggagac cgtgcagcgg ctgctgcccg tgctgtgcca ggcccacggc
     1801 ctgacacctc agcaggtggt ggccattgct agcaacatcg gaggcaagca ggccctggaa
     1861 accgtgcaaa gactgctgcc cgtgctgtgc caggcccacg gcctcacacc cgagcaagtg
     1921 gtggccatcg ctagcaacaa cggcggcaag caggctctcg agacagtcca agctctgctg
     1981 ccagtgctgt gccaagctca cggcctgaca cccgagcagg tggtcgccat cgcctcccac
     2041 gatggcggca aacaggctct ggaaacagtg caggccctgc tgcctgtgct gtgccaggcc
     2101 cacggcctga cccctgagca ggtggtggcc atcgccagca atggcggagg caagcaggcc
     2161 ctggaaaccg tgcaggctct gctgcctgtg ctgtgtcaag cccatggcct gacccctgag
     2221 caggtggtgg ctatcgcctc taatggcgga ggcaagcagg ccctggaaac cgtgcagaga
     2281 ctgctgcctg tgctgtgcca ggcccatgga ctgacccctg agcaggttgt tgcaatcgcc
     2341 agccacgacg gcggcaaaca agccctggaa accgtgcagg ccctgctgcc cgtgctgtgc
     2401 caggcccacg gactgacccc acagcaggtt gtggccatcg ccagcaacat cggcggcaag
     2461 caggccctgg aaacagtgca gcggctgctg cctgtgctgt gtcaggccca cggcctgacc
     2521 cctcaacagg tggtggccat cgccagccac gacggcggca agcaggctct tgagaccgtg
     2581 cagagactgc tgcccgtgct gtgccaggcc cacggactga cacctcagca ggtggtggcc
     2641 atcgcctccc acgacggcgg gagacctgcc ctcgaaagca ttgtggctca gctgagccgt
     2701 ccagacccgg cgctggcggc tctgaccaac gatcacctgg tggcgctggc ttgcctgggc
     2761 ggtcgtccgg ccctggatgc cgtgaagaaa ggcctgggtg gatccggcag ctacgccctg
     2821 ggtccgtatc agattagcgc cccgcagctg ccagcttaca atggtcagac cgtgggtacc
     2881 ttctactatg tgaacgacgc gggcggtctg gagagcaagg tgtttagcag cggcggtcca
     2941 accccgtacc caaactatgc caatgccggt catgtggccg gtcagagcgc cctgttcatg
     3001 cgtgataacg gcatcagcga gggtctggtg ttccacaaca acccggaagg cacctgcggt
     3061 ttttgcgtga acatgaccga gaccctgctg ccggaaaacg cgaaaatgac cgtggtgccg
     3121 ccggaaggtt ctggcggctc aactaatctg agcgacatca ttgagaagga gactgggaaa
     3181 cagctggtca ttcaggagtc catcctgatg ctgcctgagg aggtggagga agtgatcggc
     3241 aacaagccag agtctgacat cctggtgcac accgcctacg acgagtccac agatgagaat
     3301 gtgatgctgc tgacctctga cgcccccgag tataagcctt gggccctggt catccaggat
     3361 tctaacggcg agaataagat caagatgctg ggaagcggag ctactaactt cagcctgctg
     3421 aagcaggctg gagacgtgga ggagaaccct ggacctatgg tgtccaaggg cgaggaagat
     3481 aacatggcca tcatcaagga gttcatgagg tttaaggtcc acatggaggg ttcagtcaat
     3541 ggccacgagt tcgagattga aggcgagggc gagggccgcc cctacgaagg gacacagacg
     3601 gcgaaattga aggtgaccaa aggcgggcca ttgcccttcg catgggacat cttgtcccct
     3661 cagtttatgt atggcagcaa ggcctatgtt aagcaccccg ctgatatccc ggactacttg
     3721 aagctgtcct ttccagaggg gtttaaatgg gagcgcgtta tgaatttcga agacggagga
     3781 gtggttacgg tgacgcagga ctcatccctg caggacggag aatttatata taaggttaag
     3841 ttgagaggca caaacttccc aagcgacggc cctgtgatgc agaagaaaac aatggggtgg
     3901 gaagcttcca gcgagcgcat gtaccccgaa gatggcgccc tcaagggcga gataaagcaa
     3961 aggctgaaac ttaaggacgg cggtcattac gacgcggagg tcaagacaac ttacaaggct
     4021 aaaaaacccg ttcagttgcc tggggcttac aatgttaata tcaaacttga catcacaagc
     4081 cacaatgaag actatacgat cgtggagcag tatgaacgag cggaaggcag gcactcaacg
     4141 ggggggatgg acgagcttta caagtgataa ggggtctttg tcctctgtac tgtctctctc
     4201 cttgccccta acccaaaaag cttcattttt ctgtgtaggc tgcacaagag ccttgattga
     4261 agatatattc tttctgaaca gtatttaagg tttccaataa aatgtacacc cctcagaact
     4321 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     4381 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4441 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4501 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4561 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4621 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4681 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     4741 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     4801 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4861 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     4921 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4981 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     5041 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     5101 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     5161 ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
     5221 ataatacgac aaggtgagga actaaaccat ggccaagcct ttgtctcaag aagaatccac
     5281 cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt
     5341 cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt
     5401 tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg
     5461 caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg
     5521 acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag
     5581 tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga
     5641 gggctaagca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca
     5701 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5761 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     5821 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5881 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5941 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     6001 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     6061 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     6121 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6181 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     6241 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6301 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6361 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6421 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6481 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6541 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6601 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6661 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6721 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6781 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6841 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6901 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6961 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7021 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7081 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     7141 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     7201 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     7261 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7321 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7381 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7441 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7501 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7561 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7621 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7681 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7741 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7801 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7861 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7921 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7981 aatgttgaat actcatactc ttcctttttc aa
//
