LOCUS       sequence-373132-        7359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eNme2-T.2-ABE8e in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7359)
  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 7359)
  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-T.2-ABE8e
FEATURES             Location/Qualifiers
     source          1..7359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(16..33)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(187..775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(267..286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(946..1737,1738..1806))
                     /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     1569..1588
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1807..1911)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1990..2009)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2181..2560
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2561..2764
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2715..2735
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2806..2825
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2806..2824
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2874..2894
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6774..6794
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6803..6820
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6843..6860)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6849..7073
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(7144..7160)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7144..7160)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7157..7179)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7168..7184
                     /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(7192..7198,7199..7216,7217..7222))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7237..7258
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
       61 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      121 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
      181 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      241 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
      301 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
      361 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
      421 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
      481 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
      541 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
      601 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
      661 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
      721 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
      781 tcctttgatc ttttctacgg ggtctgacac tcagtggaac gaaaactcac gttaagggat
      841 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
      901 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
      961 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     1021 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     1081 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     1141 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     1201 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     1261 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     1321 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     1381 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     1441 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     1501 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     1561 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     1621 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     1681 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     1741 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     1801 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     1861 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     1921 ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc
     1981 gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     2041 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     2101 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     2161 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
     2221 ggggtcatta gttcatagcc catatattga gttccgcgtt acataactta cggtaaatgg
     2281 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     2341 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     2401 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     2461 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     2521 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     2581 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     2641 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     2701 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     2761 agctggttta gtgaaccgtc agatccgcta gagatccgcg gccgctaata cgactcacta
     2821 tagggagagc cgccaccatg aaacggacag ccgacggaag cgagttcgag tcaccaaaga
     2881 agaagcggaa agtctctgag gtggagtttt cccacgagta ctggatgaga catgccctga
     2941 ccctggccaa gagggcacgg gatgagaggg aggtgcctgt gggagccgtg ctggtgctga
     3001 acaatagagt gatcggcgag ggctggaaca gagccatcgg cctgcacgac ccaacagccc
     3061 atgccgaaat tatggccctg agacagggcg gcctggtcat gcagaactac agactgattg
     3121 acgccaccct gtacgtgaca ttcgagcctt gcgtgatgtg cgccggcgcc atgatccact
     3181 ctaggatcgg ccgcgtggtg tttggcgtga ggaactcaaa aagaggcgcc gcaggctccc
     3241 tgatgaacgt gctgaactac cccggcatga atcaccgcgt cgaaattacc gagggaatcc
     3301 tggcagatga atgtgccgcc ctgctgtgcg atttctatcg gatgcctaga caggtgttca
     3361 atgctcagaa gaaggcccag agctccatca actctggagg atctagcgga ggatcctctg
     3421 gcagcgagac accaggaaca agcgagtcag caacaccaga gagcagtggc ggcagcagcg
     3481 gcggcagcgc agcattcaag cctaacccaa tcaattacat cctgggactg gcaatcggaa
     3541 tcgcatccgt gggatgggct atggtggaga tcgacgagga ggagaatcct atccggctga
     3601 tcgatctggg cgtgagagtg tttaagaggg ccgaggtgcc aaagaccggc gattctctgg
     3661 ctatggcccg gaaactggca cggagcatga ggcgcctgac acggagaagg gcacacaggc
     3721 tgctgagggc acgccggctg ctgaagagag agggcgtgct gcaggcagca gacttcgatg
     3781 agaatggcct gatcaagagc ctgccgaaca ccccctggca gctgagagca accgccctgg
     3841 acaggaagct ggcaccactg gagtggtctg ccgtgctgct gcacttgatc aagcaccgcg
     3901 gctacctgag ccagcggaag aacgagggag agacagcaaa caagaagctg ggcgccctgc
     3961 tgaagggagt ggccaacaat gcccacgccc tgcagaccgg cgatttcagg acacctgccg
     4021 agctggccct gaataagttt gagaaggagt ccggccacat cagaaaccag aggggcgact
     4081 atagccacac cttctcccgc aaggatctgc aggccgagct gatcctgctg ttcgagaagc
     4141 agaaggattt tggcaatcca cacgtgagcg gaggcctgaa ggagggaatc gagactctgc
     4201 tgatgacaca gaggcctgcc ctgtccggcg acgcagtgca gaagatgctg ggacactgca
     4261 ccttcgagcc tgcagagcca aaggccgcca agaacaccta cacagccgag cggtttatct
     4321 ggctgacaaa gctgaacaat ctgagaatcc tggagcaggg atccgagagg ccactgaccg
     4381 acacagagag ggccaccctg atggatgagc cttaccggaa gtctaagctg acatatgccc
     4441 aggccagaaa gctgctgggc ctggaggaca ccgccttctt taagggcctg agatacggca
     4501 aggataatgc cgaggcctcc acactgatgg agatgaaggc ctatcacgcc atctctcgcg
     4561 ccctggagaa ggagggcctg aaggacaaga agtcccccct gaacctgagc tccgagctgc
     4621 aggatgagat cggcaccgcc ttctctctgt ttaagaccga cgaggatatc gcaggccgcc
     4681 tgaaggacag ggtgcagccc gagatcctgg aggccctgct gaagcacatc tctttcgata
     4741 agtttgtgca gatcagcctg aaggccctga gaaggatcgt gccactgatg gagcagggca
     4801 agcggtacga cgaggcctgc gccgagatct acggcgatcg ctatggcaag aaaaacacag
     4861 agaagaagat ctatctgccc cctatccctg ccgacgagat cagaaatcct gtggtgctga
     4921 gggccctgtc ccaggcaaga aaagtgatca acggagtggt gcgccggtac ggatctccag
     4981 cccggatcca catcgagacc gccagagaag tgggcaagag cttcaaggac cggaaggaga
     5041 tcgagaagag acaggaggag aatcgcaagg atcgggagaa ggccgccgcc aagtttaggg
     5101 agtacttccc taactttgtg ggcgagccaa agtctaagga catcctgaaa ctgcgcctgt
     5161 acgagcagca gcacggcaag tgtctgtata gcggcaagga gatcaatctg gtgcggctga
     5221 acgagaaggg ctatgtggag atcgatcacg ccctgccttt ctccagaacc tgggacgatt
     5281 cttttaacaa taaggtgctg gtgctgggca gcgagaacca gaataagggc aatcagacac
     5341 catacgagta tttcaatggc aaggacaact ccagggagtg gcaggagttc aaggcccgcg
     5401 tggagacctc tagatttccc aggagcaaga agcagcggat cctgctgcag aagttcgacg
     5461 aggatggctt taaggagtgc aacctgaatg acaccagata cgtgagccgg ttcctgtgcc
     5521 agtttgtggc cgatcacatc ctgctgaccg gcaagggcaa gagaagggtg ttcgcctcta
     5581 atggccaaat cacaaacctg ctgaggggat tttggggact gaggaaggtg cgggcagaga
     5641 atgccagaca ccactcactg gatgcagtgg tggtggcatg cagcaccgtg gcaatgcagc
     5701 agaagatcac aagattcgtg aggtataagg agatgaacgc ctttgacggc aagaccatcg
     5761 ataaggagac aggcaaggtg ctgcaccaga ggacccactt cccccagcct tgggagttct
     5821 ttgcccagga agtgatgatc cgggtgttcg gcaagccaga cggcaagcct gagtttgagg
     5881 aggccgatac cccagagaag ctgaggacac tgctggcaga gaagctgtct atcaggccag
     5941 aggcagtgca cgagtacgtg accccactgt tcgtgtccag ggcacccaat cggaagatgt
     6001 ctggcgccca caaggccaca ctgagaagcg ccaagaggtt tgtgaagcac aacgagaaga
     6061 tctccgtgaa gagagtgtgg ctgaccgaga tcaagctggc cgatctggag aacatggtga
     6121 attacaagaa cggcagggag atcgagctgt atgaggccct gaaggcaagg ctggaggcct
     6181 acggaggaaa tgccaagcag gccttcgacc caaaggataa ccccttttat aagaagggag
     6241 gacagctggt gaaggccgtg cgggtggaga agacccagaa gagcggcgtg ctgctgaata
     6301 agaggaacgc ctacacaatc gccgacaatg gcgatagggt gagagtggac gtgttctgta
     6361 aggtggataa gaagggcaag aatcagtact ttatcgtgcc tatctatgcc tggcaggtgg
     6421 ccgagaacat cctgccagac atcgattgca agggttacag aatcgacgat agctatacat
     6481 tctgtttttc cctgcacagg tatgacctga tcgccttcca gaaggatgag aagtccaagg
     6541 tggagtttgc ctactatatc aattgcaacg cctctaacgg caatttctac ctggcctggc
     6601 acgataaggg cagcaaggag cagcagtttt gcatctccac ccagaatctg gtactgatcc
     6661 agaagtatca ggtgaacgag ctgggcaagg agatcaggcc atgtcggatg aagaagcgcc
     6721 cacccgtgcg gtctggcggc tcaaaaagaa ccgccgacgg cagcgaattc gagcccaaga
     6781 agaagaggaa agtctaaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc
     6841 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     6901 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     6961 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     7021 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     7081 ggcggaaaga accagctggg gctcgatacc gtcgacctct agctagagct tggcgtaatc
     7141 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     7201 agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac tcacattaat
     7261 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     7321 aatcggccaa cgcgcgggga gaggcggttt gcgtattgg
//
