LOCUS       sequence-373129-        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"
     enhancer        128..507
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        508..711
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     662..682
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     753..772
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        753..771
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             821..841
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4721..4741
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4750..4767
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4790..4807)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4796..5020
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5091..5107)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5091..5107)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5104..5126)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5115..5131
                     /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(5139..5145,5146..5163,5164..5169))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5184..5205
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5322..5339)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5493..6081)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5573..5592)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6252..7043,7044..7112))
                     /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     6875..6894
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7113..7217)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7296..7315)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
       61 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
      121 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
      181 catagcccat atattgagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      241 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      301 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      361 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      421 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      481 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      541 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      601 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      661 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg
      721 aaccgtcaga tccgctagag atccgcggcc gctaatacga ctcactatag ggagagccgc
      781 caccatgaaa cggacagccg acggaagcga gttcgagtca ccaaagaaga agcggaaagt
      841 ctctgaggtg gagttttccc acgagtactg gatgagacat gccctgaccc tggccaagag
      901 ggcacgggat gagagggagg tgcctgtggg agccgtgctg gtgctgaaca atagagtgat
      961 cggcgagggc tggaacagag ccatcggcct gcacgaccca acagcccatg ccgaaattat
     1021 ggccctgaga cagggcggcc tggtcatgca gaactacaga ctgattgacg ccaccctgta
     1081 cgtgacattc gagccttgcg tgatgtgcgc cggcgccatg atccactcta ggatcggccg
     1141 cgtggtgttt ggcgtgagga actcaaaaag aggcgccgca ggctccctga tgaacgtgct
     1201 gaactacccc ggcatgaatc accgcgtcga aattaccgag ggaatcctgg cagatgaatg
     1261 tgccgccctg ctgtgcgatt tctatcggat gcctagacag gtgttcaatg ctcagaagaa
     1321 ggcccagagc tccatcaact ctggaggatc tagcggagga tcctctggca gcgagacacc
     1381 aggaacaagc gagtcagcaa caccagagag cagtggcggc agcagcggcg gcagcgcagc
     1441 attcaagtca aacccaatca attacatcct gggactggca atcggaatcg catccgtggg
     1501 atgggctatg gtggagatcg acgaggaggg gaatcctatc cggctgatcg atctgggcgt
     1561 gagagtgttt gagagggccg aggtgccaaa gaccggcgat tctctggcta tggcccggag
     1621 actggcacgg agcgtgaggc gcctgacacg gagaagggca cacaggctgc tgagggcacg
     1681 ccggctgctg aagagagagg gcgtgctgca ggcagcagac ttcgatgaga atggcctgat
     1741 cacgagcttg ccaaacaccc cctggcagct gagagcagcc gccctggaca ggaagctgac
     1801 accactggag tggtctgccg tgctgctgca cctgatcaag caccgcggct acctgagcca
     1861 gcggaagaac gagggagaga cagcagccaa ggagctgggc gccctgctga agggagtggc
     1921 caacaatgcc cacgccctgc agaccggcga tttcaggaca cctgccgagc tggccctgaa
     1981 taagtttgag aaggagtccg gccacatcag aaaccagagg ggcgactata gccacacctt
     2041 ctcccgcaag gatctgcagg ccgagctgat cctgctgttc gagaagcaga aggagtttgg
     2101 caatccacac gtgagcggag gcctgaagga gggaatcgag accctgctga tgacacagag
     2161 gcctgccctg tccggcgacg cagtgcagaa gatgctgggg cactgcaccc tcgagcctac
     2221 agagccaaag gccgccaaga acacctacac agccgagcgg tttatctggc tgacaaagct
     2281 gaacaatctg agaatcctgg agcagggatc cgagaggcca ctgaccgaca cagagaggtc
     2341 caccctgatg gatgagcctt accggaagtc taaactgaca tatgcccagg ccagaaagct
     2401 gctgggcctg gaggacaccg ccttctttaa gggcctgaga tacggcaagg ataatgccga
     2461 ggcctccaca ctgatggaga tgaaggccta tcacgccatc tctcgcgccc tggagaagga
     2521 gggcctgaag gacaagaagt cccccctgaa cctgagctcc gagctgcagg atgagatcgg
     2581 caccgccttc tctctgttta agaccgacga ggatatcaca ggccgcctga aggacagggt
     2641 gcagcctgag atcctggagg ccctgctgaa gcacatctct ttcgataagt ttgtgcagat
     2701 cagcctgaag gccctgagaa ggatcgtgcc actgatggag cagggcaagc ggtacgacga
     2761 ggcctgcgcc gagatctacg gcgttcacta tggcaagaag aacacagagg agaagatcta
     2821 tctgccccct atccctgccg acgagatcag aaatcctgtg gtgctgaggg ccctgtccca
     2881 ggcaagaaaa gtgatcaacg gagtggtgcg ccggtacgga tctccagccc ggatccacat
     2941 cgagaccgcc agagaagtgg gcaagagctt caaggaccgg aaggagatcg cgaagagaca
     3001 ggaggagaat cgcaaggatc gggagaaggc cgccgccaag tttagggagt acttccctaa
     3061 ctttgtgggc gagccaaagt ctaaggacat cctgaagctg cgcctgtacg agcagcagca
     3121 cggcaagtgt ctgtatagcg gcaaagagat caatctggtg cggctgaacg agaagggcta
     3181 tgtggagatc gatcacgccc tgcctttctc cagaacctgg gacgattctt ttaacaataa
     3241 ggtgctggtg ctgggcagcg agaaccagaa taagggcaat cagacaccat acgagtattt
     3301 caatggcaag gacaactcca gggagtggca ggagttcaag gcccgcgtgg agacctctag
     3361 atttcccagt agcaagaagc agcggatcct gctgcagaag ttcgacgagg atggctttaa
     3421 ggagtgcaac ctgaatgaca ccagatacgt gaaccggttc ctgtgccagt ttgtggccga
     3481 tcacatcctg ctgaccggca agggcaagag aagggtggtc gcctctaatg gccagatcac
     3541 aaacctgctg agggggtttt ggagactgag gaaggtgcgg gcagagaatg acagacacca
     3601 cgcactggat gcagtggtgg tggcatgcag caccgtggca atgcagcaga agatcacaag
     3661 attcgtgagg tataaggaga tgaacgcctt tgacggcaag accgtcgata aggagacagg
     3721 caaggtgctg taccagaaga cccacttccc ccagccttgg gagttctttg cccaggaagt
     3781 tatgatccgg gtgttcggca agccagacgg caagcctgag tttgaggagg ccgatacccc
     3841 agagaagctg aggacactgc tggcagagaa gctgtctagc aggccagagg cagtgcacga
     3901 gtacgtgacc ccgctgttcg tgtccagggc acccaatcgg aagatgtctg gcgcccacaa
     3961 ggacacactg agaagcgcca agaggtttgt gaagcacaac gagaagatct ccgtgaagag
     4021 agtgtggctg accgagatca agctggccga tctggagaac atggtgaatt acaagaacgg
     4081 cagggagatc gagctgtatg aggccctgaa ggcaaggctg gaggcctacg gaggaaatgc
     4141 caagcaggcc ttcgacccaa aggataaccc cttttataag aagggaggac agctggtgaa
     4201 ggccgtgcgg gtggagaaga cccagaagag cggcgtgctg ctgaataaga agaacgccta
     4261 cacaatcgcc gacaatggtg atatggtgag agtggacgtg ttctgtaagg tggataagaa
     4321 gggcaagaat cagtacttta tcgtgcctat ctatgcctgg caggtggccg agaacatcct
     4381 gccagacatc gattgcaagg gctacagaat cgacgatagc tatacattct gtttttccct
     4441 gcacaagtat gacctgatcg ccttccagaa ggatgagaag tccaaggtgg agtttgccta
     4501 ctatatcaat tgcgactcct ctagcggcgg gttctacctg gcctggcacg ataagggcag
     4561 cagggagcag cggtttcgca tctccaccca gaatctggcg ctgatccaga agtatcaggt
     4621 gaacgagctg ggcaaggaga tcaggccatg tcggctgaag aagcgcccac ccgtgcggtc
     4681 tggcggctca aaaagaaccg ccgacggcag cgaattcgag cccaagaaga agaggaaagt
     4741 ctaaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg
     4801 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     4861 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     4921 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     4981 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     5041 agctggggct cgataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     5101 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5161 aagtgtaaag cctagggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5221 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5281 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5341 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5401 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5461 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5521 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5581 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5641 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5701 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5761 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5821 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5881 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5941 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6001 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6061 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacactcag
     6121 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6181 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6241 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6301 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6361 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6421 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6481 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6541 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6601 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6661 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6721 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6781 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6841 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6901 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6961 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7021 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7081 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7141 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7201 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     7261 gatcgatctc ccgatcccct agggtcgact ctcagtacaa tctgctctga tgccgcatag
     7321 ttaagccagt atctgctccc tgcttgtgtg ttggaggtc
//
