LOCUS       sequence-373131-        7359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eNme2-T.1-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.1-ABE8e
FEATURES             Location/Qualifiers
     source          1..7359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        91..470
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        471..674
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     625..645
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     716..735
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        716..734
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             784..804
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4684..4704
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4713..4730
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4753..4770)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4759..4983
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5054..5070)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5054..5070)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5067..5089)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5078..5094
                     /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(5102..5108,5109..5126,5127..5132))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5147..5168
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5285..5302)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5456..6044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5536..5555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6215..7006,7007..7075))
                     /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     6838..6857
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7076..7180)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7259..7278)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
       61 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
      121 aatcaattac ggggtcatta gttcatagcc catatattga gttccgcgtt acataactta
      181 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      241 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      301 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      361 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      421 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      481 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      541 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      601 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      661 atataagcag agctggttta gtgaaccgtc agatccgcta gagatccgcg gccgctaata
      721 cgactcacta tagggagagc cgccaccatg aaacggacag ccgacggaag cgagttcgag
      781 tcaccaaaga agaagcggaa agtctctgag gtggagtttt cccacgagta ctggatgaga
      841 catgccctga ccctggccaa gagggcacgg gatgagaggg aggtgcctgt gggagccgtg
      901 ctggtgctga acaatagagt gatcggcgag ggctggaaca gagccatcgg cctgcacgac
      961 ccaacagccc atgccgaaat tatggccctg agacagggcg gcctggtcat gcagaactac
     1021 agactgattg acgccaccct gtacgtgaca ttcgagcctt gcgtgatgtg cgccggcgcc
     1081 atgatccact ctaggatcgg ccgcgtggtg tttggcgtga ggaactcaaa aagaggcgcc
     1141 gcaggctccc tgatgaacgt gctgaactac cccggcatga atcaccgcgt cgaaattacc
     1201 gagggaatcc tggcagatga atgtgccgcc ctgctgtgcg atttctatcg gatgcctaga
     1261 caggtgttca atgctcagaa gaaggcccag agctccatca actctggagg atctagcgga
     1321 ggatcctctg gcagcgagac accaggaaca agcgagtcag caacaccaga gagcagtggc
     1381 ggcagcagcg gcggcagcgc agcattcaag cctaacccaa tcaattacat cctgggactg
     1441 gcaatcggaa tcgcatccgt gggatgggct atggtggaga tcgacgagga ggagaatcct
     1501 atccggctga tcgatctggg cgtgagagtg tttaagaggg ccgaggtgcc aaagaccggc
     1561 gattctctgg ctatggcccg gagactggca cggagcatga ggcgcctgac acggagaagg
     1621 gcacacaggc tgctgagggc acgccggctg ctgaagagag agggcgtgct gcaggcagca
     1681 gacttcgatg agaatggcct gatcaagagc ctgccgaaca ccccttggca gctgagagca
     1741 gccgccctgg acaggaagct ggcaccactg gagtggtctg ccgtgctgct gcacttgatc
     1801 aagcaccgcg gctacctgag ccagcggaag aacgagggag agacagcagg caagaagctg
     1861 ggcgccctgc tgaagggagt ggccaacaat gcccacgccc tgcagaccgg cgatttcagg
     1921 acacctgccg agctggccct gaataagttt gagaaggagt ccgggcacat caggaaccag
     1981 aggggcgact atagccacac cttctcccgc aaggatctgc aggccgagct gatcctgctg
     2041 ttcgagaagc agaaggagtt tggcaatcca cacgtgagcg gaggcctgaa ggagggaatc
     2101 gagaccctgc tgatgacaca gaggcctgcc ctgtccggcg acgcagtgca gaagatgctg
     2161 ggacactgca ccttcgagcc tgcagagcca aaggccgcta agaacaccta cacagccgag
     2221 cggtttatct ggctgacaaa gctgaacaat ctgagaatcc tggagcaggg atccgagagg
     2281 ccactgaccg acacagagag ggccaccctg atggatgagc cttaccggaa gtctaagctg
     2341 acatatgccc aggccagaaa gctgctgggc ctggaggaca ccgccttctt taagggcctg
     2401 agatacggca aggataatgc cgaggcctcc acactgatgg agatgaaggc ctatcacgcc
     2461 atctctcgcg ccctggagaa ggagggcctg aaggacaaga agtcccccct gaacctgagc
     2521 tccgagctgc aggatgagat cggcaccgcc ttctctctgt ttaagaccga cgaggatatc
     2581 gcaggccgcc tgaaggacag ggtgcagcct gagatcctgg aggccctgct gaagaacatc
     2641 tctttcgata agtttgtgca gatcagcctg aagtccctga gaaggatcgt gccactgatg
     2701 gagcagggca agcggtacga cgaggcctgc gccgagatct acggcgatcg ctatggcaag
     2761 aagaacacag aggcgaagat ctatctgccc cctatccctg ccgacgagat cagaaatcct
     2821 gtggtgctga gggccctgtc ccagacaaga aaagtgatca acggagtggt gcgccggtac
     2881 ggatctccag cccggatcca catcgagacc gccagagaag tgggcaagag cttcaaggac
     2941 cggaaggaga tcgagaagag acaggaggag aatcgcaagg atcgggagaa ggccgccgcc
     3001 aaatttaggg agtacttccc taattttgtg ggcgagccaa agtctaagga catcctgaag
     3061 ctgcgcctgt acgagcagca gcacggcaag tgtctgtata gcggcaagga gatcaatctg
     3121 gtgcggctga acgagaaggg ctatgtggag atcgatcacg ccctgccttt ctccagaacc
     3181 tgggacgatt cttttaacaa taaggtgctg gtgctgggca gcgagaacca gaataagggc
     3241 aatcagacac catacgagta tttcaatggc aaggacaacc ccagggagtg gcaggagttc
     3301 aaggcccgcg tggagacctc tagatttccc aggagcaaga agcagcggat cctgctgcag
     3361 aaattcgacg aggatggctt taaggagtgc aacctgaatg acaccagata cgtgagccgg
     3421 ttcttgtgcc agtttgtggc cgatcacatc ctgctgaccg gcaagggcaa gagaagggtg
     3481 ttcgcctcta atggccagat cacaaacctg ctgaggggat tttggggact gaggaaggtg
     3541 cgggcagaga atgccagaca ccacgcactg gatgcagtgg tagtggcatg cagcaccgtg
     3601 gcaatgcagc agaagatcac aagattcgtg aggtataagg agatgaacgc ctttgacggc
     3661 aagaccatcg ataaggagac aggcaaggcg ctgtaccaga agacccgctt cccccagcct
     3721 tgggagttct ttgcccagga agtgatgatc cgggtgttcg gcaagccaga cggcaagcct
     3781 gagtttgagg aggccgatac cccagagaag ctgaggacac tgctggcaga gaagctgtct
     3841 agcaggccag aggcagcgca cgagtacgtg accccactgt tcgtgtccag ggcacccaat
     3901 cggaagatgt ctggcgccca caaggccaca ctgagaagcg ccaagaggtt tgtgaagcac
     3961 aacgagaagg tctccgtgaa gagagtgttg ctgaccgaga tcaagctggc cgatctggag
     4021 aacatggtga attacaagaa cggcagggag atcgagctgt atgaggccct gaaggcaagg
     4081 ctggaggcct acggaggaaa tgccaagcag gccttcgacc caaaggataa ccccttttat
     4141 aagaagggag gacagctggt gaaggccgtg cgggtggaga agacccagga gagcggcgtg
     4201 ctgctgaata agaagaacgc ctacacaatc gccgacaatg gcgatagggt gagagtggac
     4261 gtgttctgta aggtggataa gaagggcaag aatcagtact ttatcgtgcc tatctatgcc
     4321 tggcaggtgg ccgagaacat cctgccagac atcgattgca agggctacag aatcgacgat
     4381 agctatacat tctgtttttc cctgcacagg tatgacctga tcgccttcca gaaggatgag
     4441 aagtccaagg tggagtttgc ctactatatc aattgcaacg cctctaacgg ctatttctac
     4501 ctggcctggc acgataaggg cagcaaggag cagcagttta gcatctccac ccagaatctg
     4561 gtgctgatcc agaagtatca ggtgagcgag ctgggcaagg agatcaggcc atgtcggctg
     4621 aagaagcgcc cacccgtgcg gtctggcggc tcaaaaagaa ccgccgacgg cagcgaattc
     4681 gagcccaaga agaagaggaa agtctaaccg gtcatcatca ccatcaccat tgagtttaaa
     4741 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     4801 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     4861 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     4921 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     4981 tggcttctga ggcggaaaga accagctggg gctcgatacc gtcgacctct agctagagct
     5041 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5101 acaacatacg agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac
     5161 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5221 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5281 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5341 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5401 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5461 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5521 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5581 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5641 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5701 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5761 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5821 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5881 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5941 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6001 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6061 tttctacggg gtctgacact cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6121 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6181 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6241 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6301 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6361 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6421 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6481 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6541 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6601 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6661 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6721 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6781 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6841 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6901 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6961 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7021 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7081 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7141 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7201 cacctgacgt cgacggatcg ggagatcgat ctcccgatcc cctagggtcg actctcagta
     7261 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     7321 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaa
//
