LOCUS       sequence-351815-        7913 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 7913)
  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 7913)
  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- T1413I-GFP
FEATURES             Location/Qualifiers
     source          1..7913
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             739..987
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             997..1053
                     /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"
     CDS             join(1054..1056,1057..1059,1060..1773)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1054..1056,1057..1059,1060..1770)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1099..1120)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1360..1379)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1707..1728
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      1970..2398
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2057..2076)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2267..2288
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2407..2427)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2412..2741
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2592..2727
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2654..2673
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2789..2836
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2855..3253
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3411..3532
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3448..3467)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3502..3521
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3581..3597)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3581..3597)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3594..3616)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3605..3621
                     /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(3629..3635,3636..3653,3654..3659))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3674..3695
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3812..3829)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3983..4571)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4063..4082)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4742..5533,5534..5602))
                     /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     5365..5384
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5603..5707)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5782..5801)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5973..6352
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6353..6556
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6507..6527
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6601..6620
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6601..6619
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6735..6761
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6765..6791
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6795..6821
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
ORIGIN
        1 gtgcagagac tgctgcccgt gctgtgccag gcccacggcc tgacacctga gcaggtggtg
       61 gctatcgcca gcaacggcgg cggcaaacaa gccctggaaa cagtgcaagc tcttctgcct
      121 gtgctgtgtc aggcccacgg actgacccct cagcaggtgg tggccatcgc cagcaacatc
      181 ggcggtaagc aggccctgga aaccgtgcag cggctgctgc ctgtgctgtg ccaggcccac
      241 ggcctgaccc ctgagcaagt ggtggccatc gctagcaatg gaggcggcaa acaggccctg
      301 gaaaccgtgc agcggctgct gcctgtgctg tgccaggccc acggcctgac cccacaacaa
      361 gtggtggcca tcgccagcaa caatggcggc aaacaggccc tggaaaccgt gcagagactg
      421 ctgcctgtgc tgtgccaggc ccacggcctg accccccagc aggtggtggc catcgcctcc
      481 aatatcggcg ggagacccgc cctggaaagc attgtggctc agctgagccg tccagacccg
      541 gccctggcgg ctctgaccaa cgatcacctg gtggcgctgg cttgcctggg cggtcgtccg
      601 gccctggatg cggtgaagaa aggcctgggt ggatccgcca ttccagtgaa gcgcggcgct
      661 accggtgaaa ccaaagtgtt tatcggtaac agcaacagcc cgaagagccc gaccaaaggc
      721 ggttgctctg gcggctcaac taatctgagc gacatcattg agaaggagac tgggaaacag
      781 ctggtcattc aggagtccat cctgatgctg cctgaggagg tggaggaagt gatcggcaac
      841 aagccagagt ctgacatcct ggtgcacacc gcctacgacg agtccacaga tgagaatgtg
      901 atgctgctga cctctgacgc ccccgagtat aagccttggg ccctggtcat ccaggattct
      961 aacggcgaga ataagatcaa gatgctggga agcggagcta ctaacttcag cctgctgaag
     1021 caggctggag acgtggagga gaaccctgga cctatggtga gcaagggcga ggagctgttc
     1081 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1141 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1201 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctatggagtg
     1261 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1321 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1381 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1441 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1501 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1561 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1621 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     1681 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1741 atcactctcg gcatggacga gctgtacaag tgataagggg tctttgtcct ctgtactgtc
     1801 tctctccttg cccctaaccc aaaaagcttc atttttctgt gtaggctgca caagagcctt
     1861 gattgaagat atattctttc tgaacagtat ttaaggtttc caataaaatg tacacccctc
     1921 agaacttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     1981 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2041 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2101 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2161 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     2221 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     2281 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     2341 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     2401 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2461 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     2521 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     2581 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2641 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     2701 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     2761 tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg
     2821 catagtataa tacgacaagg tgaggaacta aaccatggcc aagcctttgt ctcaagaaga
     2881 atccaccctc attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta
     2941 cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata
     3001 tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc
     3061 agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc
     3121 ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa
     3181 ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt
     3241 gtgggagggc taagcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg
     3301 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     3361 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     3421 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3481 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     3541 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3601 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3661 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3721 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3781 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3841 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3901 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3961 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4021 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4081 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4141 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4201 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4261 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4321 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatata ggcggtgcta
     4381 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4441 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4501 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4561 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4621 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4681 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4741 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4801 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4861 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4921 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4981 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5041 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5101 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5161 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5221 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5281 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5341 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5401 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5461 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5521 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5581 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5641 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5701 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     5761 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     5821 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     5881 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     5941 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
     6001 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     6061 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     6121 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     6181 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     6241 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     6301 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     6361 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     6421 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     6481 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     6541 ctatataagc agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat
     6601 taatacgact cactataggg agacccaagc tggctagcac catggccctg tcccgtgcgg
     6661 tttgtggcac ctcccgtcaa ctggctccgg ttctgggtta tctgggttcc cgtcaaaaac
     6721 actccctgcc ggactacccg tatgatgttc cggattacgc tggctaccca tacgacgtcc
     6781 cagactacgc tggctaccca tacgacgtcc cagactacgc tatggacatc gcggatctgc
     6841 gtaccctggg ttacagccag cagcagcagg agaagatcaa gccgaaggtg cgcagcaccg
     6901 tggctcagca ccacgaagcc ctggtgggcc acggtttcac ccacgctcac attgtggccc
     6961 tgagccagca cccagccgcg ctgggcaccg tggccgtgaa atatcaggat atgattgctg
     7021 ccctgccaga ggccacccat gaagctattg tgggcgtggg caagcagtgg agcggtgctc
     7081 gtgcgctgga ggcgctgctg accgtggctg gtgaactgcg tggtccgccg ctgcagctgg
     7141 acaccggtca gctgctgaaa atcgcgaaac gtggcggtgt gaccgcggtg gaagccgtgc
     7201 atgcttggcg taatgctctg accggtgcgc cgctgaacct cacacccgaa caggttgttg
     7261 ctatcgccag caacaacgga ggaaagcagg cccttgagac agtgcaggcc ctgctccccg
     7321 tgctgtgcca ggcccacgga ctgacccctc aacaggtggt tgctatcgcc tccaacaacg
     7381 gcggcaagca ggccctggaa accgtgcaga ggctgctgcc cgtgctgtgt caggcccacg
     7441 gcctgacccc tcagcaggtc gtggccatcg ccagcaacaa cggcggaaag caggcacttg
     7501 aaaccgtcca aagactgctg cccgtgctgt gccaggccca tggactgaca cccgagcagg
     7561 tggtggccat tgccagccac gacggcggaa agcaagctct ggagaccgtt caagctctgc
     7621 tgccagtgct gtgccaggcc cacggactga caccagagca ggtggtggct atcgcctcta
     7681 acaacggcgg caagcaggcc cttgaaaccg tgcaggctct gctgcctgtg ctgtgccagg
     7741 ctcacggcct gacacctgag caggtcgtgg ccatcgcctc taatatcggc ggcaagcagg
     7801 ccctggagac agtgcaggct ctgctgcctg ttctgtgtca ggcccacggc ctgacccctg
     7861 agcaggtggt ggccatcgcc agcaacggcg gcggcaagca ggccctcgag aca
//
