LOCUS       sequence-374493-        8103 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eNme2-C-BE4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8103)
  AUTHORS   Huang TP, Heins ZJ, Miller SM, Wong BG, Balivada PA, Wang T, Khalil 
            AS, Liu DR
  TITLE     High-throughput continuous evolution of compact Cas9 variants 
            targeting single-nucleotide-pyrimidine PAMs.
  JOURNAL   Nat Biotechnol. 2022 Sep 8. pii: 10.1038/s41587-022-01410-2. doi: 
            10.1038/s41587-022-01410-2.
  PUBMED    36076084
REFERENCE   2  (bases 1 to 8103)
  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: "Nat 
            Biotechnol. 2022 Sep 8. pii: 10.1038/s41587-022-01410-2. doi: 
            10.1038/s41587-022-01410-2."
COMMENT     Sequence Label: pCMV-eNme2-C-BE4max
FEATURES             Location/Qualifiers
     source          1..8103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        259..462
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     413..433
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     504..523
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        504..522
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             572..592
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             593..1276
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             4646..4894
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4925..5173
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5216..5236
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5245..5262
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5285..5302)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5291..5515
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5586..5602)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5586..5602)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5599..5621)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5610..5626
                     /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(5634..5640,5641..5658,5659..5664))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5679..5700
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5817..5834)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5988..6576)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6068..6087)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6747..7538,7539..7607))
                     /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     7370..7389
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7608..7712)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7791..7810)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7982..8103,1..258)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
       61 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      121 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      181 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      241 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      301 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      361 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      421 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
      481 atccgctaga gatccgcggc cgctaatacg actcactata gggagagccg ccaccatgaa
      541 acggacagcc gacggaagcg agttcgagtc accaaagaag aagcggaaag tctcctcaga
      601 gactgggcct gtcgccgtcg atccaaccct gcgccgccgg attgaacctc acgagtttga
      661 agtgttcttt gacccccggg agctgagaaa ggagacatgc ctgctgtacg agatcaactg
      721 gggaggcagg cactccatct ggaggcacac ctctcagaac acaaataagc acgtggaggt
      781 gaacttcatc gagaagttta ccacagagcg gtacttctgc cccaatacca gatgtagcat
      841 cacatggttt ctgagctggt ccccttgcgg agagtgtagc agggccatca ccgagttcct
      901 gtccagatat ccacacgtga cactgtttat ctacatcgcc aggctgtatc accacgcaga
      961 cccaaggaat aggcagggcc tgcgcgatct gatcagctcc ggcgtgacca tccagatcat
     1021 gacagagcag gagtccggct actgctggcg gaacttcgtg aattattctc ctagcaacga
     1081 ggcccactgg cctaggtacc cacacctgtg ggtgcgcctg tacgtgctgg agctgtattg
     1141 catcatcctg ggcctgcccc cttgtctgaa tatcctgcgg agaaagcagc cccagctgac
     1201 cttctttaca atcgccctgc agtcttgtca ctatcagagg ctgccacccc acatcctgtg
     1261 ggccacaggc ctgaagtctg gaggatctag cggaggatcc tctggcagcg agacaccagg
     1321 aacaagcgag tcagcaacac cagagagcag tggcggcagc agcggcggca gcgcagcatt
     1381 caagtcaaac ccaatcaatt acatcctggg actggcaatc ggaatcgcat ccgtgggatg
     1441 ggctatggtg gagatcgacg aggaggggaa tcctatccgg ctgatcgatc tgggcgtgag
     1501 agtgtttgag agggccgagg tgccaaagac cggcgattct ctggctatgg cccggagact
     1561 ggcacggagc gtgaggcgcc tgacacggag aagggcacac aggctgctga gggcacgccg
     1621 gctgctgaag agagagggcg tgctgcaggc agcagacttc gatgagaatg gcctgatcac
     1681 gagcttgcca aacaccccct ggcagctgag agcagccgcc ctggacagga agctgacacc
     1741 actggagtgg tctgccgtgc tgctgcacct gatcaagcac cgcggctacc tgagccagcg
     1801 gaagaacgag ggagagacag cagccaagga gctgggcgcc ctgctgaagg gagtggccaa
     1861 caatgcccac gccctgcaga ccggcgattt caggacacct gccgagctgg ccctgaataa
     1921 gtttgagaag gagtccggcc acatcagaaa ccagaggggc gactatagcc acaccttctc
     1981 ccgcaaggat ctgcaggccg agctgatcct gctgttcgag aagcagaagg agtttggcaa
     2041 tccacacgtg agcggaggcc tgaaggaggg aatcgagacc ctgctgatga cacagaggcc
     2101 tgccctgtcc ggcgacgcag tgcagaagat gctggggcac tgcaccctcg agcctacaga
     2161 gccaaaggcc gccaagaaca cctacacagc cgagcggttt atctggctga caaagctgaa
     2221 caatctgaga atcctggagc agggatccga gaggccactg accgacacag agaggtccac
     2281 cctgatggat gagccttacc ggaagtctaa actgacatat gcccaggcca gaaagctgct
     2341 gggcctggag gacaccgcct tctttaaggg cctgagatac ggcaaggata atgccgaggc
     2401 ctccacactg atggagatga aggcctatca cgccatctct cgcgccctgg agaaggaggg
     2461 cctgaaggac aagaagtccc ccctgaacct gagctccgag ctgcaggatg agatcggcac
     2521 cgccttctct ctgtttaaga ccgacgagga tatcacaggc cgcctgaagg acagggtgca
     2581 gcctgagatc ctggaggccc tgctgaagca catctctttc gataagtttg tgcagatcag
     2641 cctgaaggcc ctgagaagga tcgtgccact gatggagcag ggcaagcggt acgacgaggc
     2701 ctgcgccgag atctacggcg ttcactatgg caagaagaac acagaggaga agatctatct
     2761 gccccctatc cctgccgacg agatcagaaa tcctgtggtg ctgagggccc tgtcccaggc
     2821 aagaaaagtg atcaacggag tggtgcgccg gtacggatct ccagcccgga tccacatcga
     2881 gaccgccaga gaagtgggca agagcttcaa ggaccggaag gagatcgcga agagacagga
     2941 ggagaatcgc aaggatcggg agaaggccgc cgccaagttt agggagtact tccctaactt
     3001 tgtgggcgag ccaaagtcta aggacatcct gaagctgcgc ctgtacgagc agcagcacgg
     3061 caagtgtctg tatagcggca aagagatcaa tctggtgcgg ctgaacgaga agggctatgt
     3121 ggagatcgat cacgccctgc ctttctccag aacctgggac gattctttta acaataaggt
     3181 gctggtgctg ggcagcgaga accagaataa gggcaatcag acaccatacg agtatttcaa
     3241 tggcaaggac aactccaggg agtggcagga gttcaaggcc cgcgtggaga cctctagatt
     3301 tcccagtagc aagaagcagc ggatcctgct gcagaagttc gacgaggatg gctttaagga
     3361 gtgcaacctg aatgacacca gatacgtgaa ccggttcctg tgccagtttg tggccgatca
     3421 catcctgctg accggcaagg gcaagagaag ggtggtcgcc tctaatggcc agatcacaaa
     3481 cctgctgagg gggttttgga gactgaggaa ggtgcgggca gagaatgaca gacaccacgc
     3541 actggatgca gtggtggtgg catgcagcac cgtggcaatg cagcagaaga tcacaagatt
     3601 cgtgaggtat aaggagatga acgcctttga cggcaagacc gtcgataagg agacaggcaa
     3661 ggtgctgtac cagaagaccc acttccccca gccttgggag ttctttgccc aggaagttat
     3721 gatccgggtg ttcggcaagc cagacggcaa gcctgagttt gaggaggccg ataccccaga
     3781 gaagctgagg acactgctgg cagagaagct gtctagcagg ccagaggcag tgcacgagta
     3841 cgtgaccccg ctgttcgtgt ccagggcacc caatcggaag atgtctggcg cccacaagga
     3901 cacactgaga agcgccaaga ggtttgtgaa gcacaacgag aagatctccg tgaagagagt
     3961 gtggctgacc gagatcaagc tggccgatct ggagaacatg gtgaattaca agaacggcag
     4021 ggagatcgag ctgtatgagg ccctgaaggc aaggctggag gcctacggag gaaatgccaa
     4081 gcaggccttc gacccaaagg ataacccctt ttataagaag ggaggacagc tggtgaaggc
     4141 cgtgcgggtg gagaagaccc agaagagcgg cgtgctgctg aataagaaga acgcctacac
     4201 aatcgccgac aatggtgata tggtgagagt ggacgtgttc tgtaaggtgg ataagaaggg
     4261 caagaatcag tactttatcg tgcctatcta tgcctggcag gtggccgaga acatcctgcc
     4321 agacatcgat tgcaagggct acagaatcga cgatagctat acattctgtt tttccctgca
     4381 caagtatgac ctgatcgcct tccagaagga tgagaagtcc aaggtggagt ttgcctacta
     4441 tatcaattgc gactcctcta gcggcgggtt ctacctggcc tggcacgata agggcagcag
     4501 ggagcagcgg tttcgcatct ccacccagaa tctggcgctg atccagaagt atcaggtgaa
     4561 cgagctgggc aaggagatca ggccatgtcg gctgaagaag cgcccacccg tgcggagcgg
     4621 tggtagcggc gggagcgggg ggagcactaa tctgagcgac atcattgaga aggagactgg
     4681 gaaacagctg gtcattcagg agtccatcct gatgctgcct gaggaggtgg aggaagtgat
     4741 cggcaacaag ccagagtctg acatcctggt gcacaccgcc tacgacgagt ccacagatga
     4801 gaatgtgatg ctgctgacct ctgacgcccc cgagtataag ccttgggccc tggtcatcca
     4861 ggattctaac ggcgagaata agatcaagat gctgagcgga ggatccggag gatctggagg
     4921 cagcaccaac ctgtctgaca tcatcgagaa ggagacaggc aagcagctgg tcatccagga
     4981 gagcatcctg atgctgcccg aagaagtcga agaagtgatc ggaaacaagc ctgagagcga
     5041 tatcctggtc cataccgcct acgacgagag taccgacgaa aatgtgatgc tgctgacatc
     5101 cgacgcccca gagtataagc cctgggctct ggtcatccag gattccaacg gagagaacaa
     5161 aatcaaaatg ctgtctggcg gctcaaaaag aaccgccgac ggcagcgaat tcgagcccaa
     5221 gaagaagagg aaagtctaac cggtcatcat caccatcacc attgagttta aacccgctga
     5281 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     5341 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     5401 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     5461 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     5521 gaggcggaaa gaaccagctg gggctcgata ccgtcgacct ctagctagag cttggcgtaa
     5581 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5641 cgagccggaa gcataaagtg taaagcctag ggtgcctaat gagtgagcta actcacatta
     5701 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5761 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5821 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5881 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5941 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6001 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6061 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6121 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6181 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6241 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6301 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6361 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6421 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6481 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6541 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6601 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6661 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6721 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6781 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6841 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6901 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6961 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7021 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7081 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7141 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7201 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7261 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7321 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7381 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7441 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7501 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7561 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7621 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7681 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7741 gtcgacggat cgggagatcg atctcccgat cccctagggt cgactctcag tacaatctgc
     7801 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     7861 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     7921 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     7981 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     8041 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     8101 aac
//
