LOCUS       sequence-471552-        7359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eNme2-C-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-C-ABE8e
FEATURES             Location/Qualifiers
     source          1..7359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(651..671)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(720..739)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(721..739)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(781..984)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(810..830)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        985..1364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1536..1555
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1634..1738
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1739..1807,1808..2599)
                     /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     complement(1957..1976)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2770..3358
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3259..3278
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3512..3529
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3646..3667
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3682..3687,3688..3705,3706..3712)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3720..3736
                     /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)."
     primer_bind     3725..3747
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3744..3760
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3744..3760
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3831..4055
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4044..4061
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4084..4101)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4110..4130)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 gcgattccga ttgccagtcc caggatgtaa ttgattgggt ttgacttgaa tgctgcgctg
       61 ccgccgctgc tgccgccact gctctctggt gttgctgact cgcttgttcc tggtgtctcg
      121 ctgccagagg atcctccgct agatcctcca gagttgatgg agctctgggc cttcttctga
      181 gcattgaaca cctgtctagg catccgatag aaatcgcaca gcagggcggc acattcatct
      241 gccaggattc cctcggtaat ttcgacgcgg tgattcatgc cggggtagtt cagcacgttc
      301 atcagggagc ctgcggcgcc tctttttgag ttcctcacgc caaacaccac gcggccgatc
      361 ctagagtgga tcatggcgcc ggcgcacatc acgcaaggct cgaatgtcac gtacagggtg
      421 gcgtcaatca gtctgtagtt ctgcatgacc aggccgccct gtctcagggc cataatttcg
      481 gcatgggctg ttgggtcgtg caggccgatg gctctgttcc agccctcgcc gatcactcta
      541 ttgttcagca ccagcacggc tcccacaggc acctccctct catcccgtgc cctcttggcc
      601 agggtcaggg catgtctcat ccagtactcg tgggaaaact ccacctcaga gactttccgc
      661 ttcttctttg gtgactcgaa ctcgcttccg tcggctgtcc gtttcatggt ggcggctctc
      721 cctatagtga gtcgtattag cggccgcgga tctctagcgg atctgacggt tcactaaacc
      781 agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc
      841 ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt
      901 gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt
      961 gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg
     1021 ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc
     1081 gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg
     1141 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac
     1201 tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg
     1261 cgggccattt accgtaagtt atgtaacgcg gaactcaata tatgggctat gaactaatga
     1321 ccccgtaatt gattactatt aataactagt caataatcaa tgtcaacgcg tatatctggc
     1381 ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc agattcttca tgcaattgtc
     1441 ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg cactactcag cgacctccaa
     1501 cacacaagca gggagcagat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     1561 agtcgaccct aggggatcgg gagatcgatc tcccgatccg tcgacgtcag gtggcacttt
     1621 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     1681 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     1741 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     1801 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     1861 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     1921 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     1981 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2041 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2101 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     2161 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     2221 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     2281 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     2341 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     2401 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     2461 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     2521 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     2581 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     2641 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     2701 caaaatccct taacgtgagt tttcgttcca ctgagtgtca gaccccgtag aaaagatcaa
     2761 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2821 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2881 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     2941 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3001 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3061 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3121 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3181 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3241 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3301 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3361 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3421 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3481 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3541 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     3601 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     3661 cactcattag gcaccctagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     3721 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcta
     3781 gctagaggtc gacggtatcg agccccagct ggttctttcc gcctcagaag ccatagagcc
     3841 caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc
     3901 caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt
     3961 ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg
     4021 gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac
     4081 tcaatggtga tggtgatgat gaccggttag actttcctct tcttcttggg ctcgaattcg
     4141 ctgccgtcgg cggttctttt tgagccgcca gaccgcacgg gtgggcgctt cttcagccga
     4201 catggcctga tctccttgcc cagctcgttc acctgatact tctggatcag cgccagattc
     4261 tgggtggaga tgcgaaaccg ctgctccctg ctgcccttat cgtgccaggc caggtagaac
     4321 ccgccgctag aggagtcgca attgatatag taggcaaact ccaccttgga cttctcatcc
     4381 ttctggaagg cgatcaggtc atacttgtgc agggaaaaac agaatgtata gctatcgtcg
     4441 attctgtagc ccttgcaatc gatgtctggc aggatgttct cggccacctg ccaggcatag
     4501 ataggcacga taaagtactg attcttgccc ttcttatcca ccttacagaa cacgtccact
     4561 ctcaccatat caccattgtc ggcgattgtg taggcgttct tcttattcag cagcacgccg
     4621 ctcttctggg tcttctccac ccgcacggcc ttcaccagct gtcctccctt cttataaaag
     4681 gggttatcct ttgggtcgaa ggcctgcttg gcatttcctc cgtaggcctc cagccttgcc
     4741 ttcagggcct catacagctc gatctccctg ccgttcttgt aattcaccat gttctccaga
     4801 tcggccagct tgatctcggt cagccacact ctcttcacgg agatcttctc gttgtgcttc
     4861 acaaacctct tggcgcttct cagtgtgtcc ttgtgggcgc cagacatctt ccgattgggt
     4921 gccctggaca cgaacagcgg ggtcacgtac tcgtgcactg cctctggcct gctagacagc
     4981 ttctctgcca gcagtgtcct cagcttctct ggggtatcgg cctcctcaaa ctcaggcttg
     5041 ccgtctggct tgccgaacac ccggatcata acttcctggg caaagaactc ccaaggctgg
     5101 gggaagtggg tcttctggta cagcaccttg cctgtctcct tatcgacggt cttgccgtca
     5161 aaggcgttca tctccttata cctcacgaat cttgtgatct tctgctgcat tgccacggtg
     5221 ctgcatgcca ccaccactgc atccagtgcg tggtgtctgt cattctctgc ccgcaccttc
     5281 ctcagtctcc aaaaccccct cagcaggttt gtgatctggc cattagaggc gaccaccctt
     5341 ctcttgccct tgccggtcag caggatgtga tcggccacaa actggcacag gaaccggttc
     5401 acgtatctgg tgtcattcag gttgcactcc ttaaagccat cctcgtcgaa cttctgcagc
     5461 aggatccgct gcttcttgct actgggaaat ctagaggtct ccacgcgggc cttgaactcc
     5521 tgccactccc tggagttgtc cttgccattg aaatactcgt atggtgtctg attgccctta
     5581 ttctggttct cgctgcccag caccagcacc ttattgttaa aagaatcgtc ccaggttctg
     5641 gagaaaggca gggcgtgatc gatctccaca tagcccttct cgttcagccg caccagattg
     5701 atctctttgc cgctatacag acacttgccg tgctgctgct cgtacaggcg cagcttcagg
     5761 atgtccttag actttggctc gcccacaaag ttagggaagt actccctaaa cttggcggcg
     5821 gccttctccc gatccttgcg attctcctcc tgtctcttcg cgatctcctt ccggtccttg
     5881 aagctcttgc ccacttctct ggcggtctcg atgtggatcc gggctggaga tccgtaccgg
     5941 cgcaccactc cgttgatcac ttttcttgcc tgggacaggg ccctcagcac cacaggattt
     6001 ctgatctcgt cggcagggat agggggcaga tagatcttct cctctgtgtt cttcttgcca
     6061 tagtgaacgc cgtagatctc ggcgcaggcc tcgtcgtacc gcttgccctg ctccatcagt
     6121 ggcacgatcc ttctcagggc cttcaggctg atctgcacaa acttatcgaa agagatgtgc
     6181 ttcagcaggg cctccaggat ctcaggctgc accctgtcct tcaggcggcc tgtgatatcc
     6241 tcgtcggtct taaacagaga gaaggcggtg ccgatctcat cctgcagctc ggagctcagg
     6301 ttcagggggg acttcttgtc cttcaggccc tccttctcca gggcgcgaga gatggcgtga
     6361 taggccttca tctccatcag tgtggaggcc tcggcattat ccttgccgta tctcaggccc
     6421 ttaaagaagg cggtgtcctc caggcccagc agctttctgg cctgggcata tgtcagttta
     6481 gacttccggt aaggctcatc catcagggtg gacctctctg tgtcggtcag tggcctctcg
     6541 gatccctgct ccaggattct cagattgttc agctttgtca gccagataaa ccgctcggct
     6601 gtgtaggtgt tcttggcggc ctttggctct gtaggctcga gggtgcagtg ccccagcatc
     6661 ttctgcactg cgtcgccgga cagggcaggc ctctgtgtca tcagcagggt ctcgattccc
     6721 tccttcaggc ctccgctcac gtgtggattg ccaaactcct tctgcttctc gaacagcagg
     6781 atcagctcgg cctgcagatc cttgcgggag aaggtgtggc tatagtcgcc cctctggttt
     6841 ctgatgtggc cggactcctt ctcaaactta ttcagggcca gctcggcagg tgtcctgaaa
     6901 tcgccggtct gcagggcgtg ggcattgttg gccactccct tcagcagggc gcccagctcc
     6961 ttggctgctg tctctccctc gttcttccgc tggctcaggt agccgcggtg cttgatcagg
     7021 tgcagcagca cggcagacca ctccagtggt gtcagcttcc tgtccagggc ggctgctctc
     7081 agctgccagg gggtgtttgg caagctcgtg atcaggccat tctcatcgaa gtctgctgcc
     7141 tgcagcacgc cctctctctt cagcagccgg cgtgccctca gcagcctgtg tgcccttctc
     7201 cgtgtcaggc gcctcacgct ccgtgccagt ctccgggcca tagccagaga atcgccggtc
     7261 tttggcacct cggccctctc aaacactctc acgcccagat cgatcagccg gataggattc
     7321 ccctcctcgt cgatctccac catagcccat cccacggat
//
