LOCUS       sequence-364078-        6765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nuclease active eNme2-C.NR in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6765)
  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 6765)
  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.NR
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        85..288
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     239..259
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     330..349
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        330..348
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             398..418
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3704..3724
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3733..3750
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3773..3790)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3779..4003
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4074..4090)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4074..4090)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4087..4109)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4098..4114
                     /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(4122..4128,4129..4146,4147..4152))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4167..4188
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4305..4322)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4476..5064)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4556..4575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5235..6026,6027..6095))
                     /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     5858..5877
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6096..6200)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6279..6298)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6470..6765,1..84)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
       61 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      121 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      181 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      241 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac
      301 cgtcagatcc gctagagatc cgcggccgct aatacgactc actataggga gagccgccac
      361 catgaaacgg acagccgacg gaagcgagtt cgagtcacca aagaagaagc ggaaagtcgc
      421 agcattcaag cctaacccaa tcaattacat cctgggactg gatatcggaa tcgcatccgt
      481 gggatgggct atggtggaga tcgacgagga ggagaatcct atccggctga tcgatctggg
      541 cgtgagagtg tttgagaggg ccgaggtgcc aaagaccggc gattctctgg ctatggcccg
      601 gagactggca cggagcgtga ggcgcctgac acggagaagg gcacacaggc tgctgagggc
      661 acgccggctg ctgaagagag agggcgtgct gcaggcagca gacttcgatg agaatggcct
      721 gatcacgagc ttgccaaaca ccccctggca gctgagagca gccgccctgg acaggaagct
      781 gacaccactg gagtggtctg ccgtgctgct gcacctgatc aagcaccgcg gctacctgag
      841 ccagcggaag aacgagggag agacagcagc caaggagctg ggcgccctgc tgaagggagt
      901 ggccaacaat gcccacgccc tgcagaccgg cgatttcagg acacctgccg agctggccct
      961 gaataagttt gagaaggagt ccggccacat cagaaaccag aggggcgact atagccacac
     1021 cttctcccgc aaggatctgc aggccgagct gatcctgctg ttcgagaagc agaaggagtt
     1081 tggcaatcca cacgtgagcg gaggcctgaa ggagggaatc gagaccctgc tgatgacaca
     1141 gaggcctgcc ctgtccggcg acgcagtgca gaagatgctg gggcactgca ccctcgagcc
     1201 tacagagcca aaggccgcca agaacaccta cacagccgag cggtttatct ggctgacaaa
     1261 gctgaacaat ctgagaatcc tggagcaggg atccgagagg ccactgaccg acacagagag
     1321 gtccaccctg atggatgagc cttaccggaa gtctaaactg acatatgccc aggccagaaa
     1381 gctgctgggc ctggaggaca ccgccttctt taagggcctg agatacggca aggataatgc
     1441 cgaggcctcc acactgatgg agatgaaggc ctatcacgcc atctctcgcg ccctggagaa
     1501 ggagggcctg aaggacaaga agtcccccct gaacctgagc tccgagctgc aggatgagat
     1561 cggcaccgcc ttctctctgt ttaagaccga cgaggatatc acaggccgcc tgaaggacag
     1621 ggtgcagcct gagatcctgg aggccctgct gaagcacatc tctttcgata agtttgtgca
     1681 gatcagcctg aaggccctga gaaggatcgt gccactgatg gagcagggca agcggtacga
     1741 cgaggcctgc gccgagatct acggcgttca ctatggcaag aagaacacag aggagaagat
     1801 ctatctgccc cctatccctg ccgacgagat cagaaatcct gtggtgctga gggccctgtc
     1861 ccaggcaaga aaagtgatca acggagtggt gcgccggtac ggatctccag cccggatcca
     1921 catcgagacc gccagagaag tgggcaagag cttcaaggac cggaaggaga tcgagaagag
     1981 acaggaggag aatcgcaagg atcgggagaa ggccgccgcc aagtttaggg agtacttccc
     2041 taactttgtg ggcgagccaa agtctaagga catcctgaag ctgcgcctgt acgagcagca
     2101 gcacggcaag tgtctgtata gcggcaagga gatcaatctg gtgcggctga acgagaaggg
     2161 ctatgtggag atcgatcacg ccctgccttt ctccagaacc tgggacgatt cttttaacaa
     2221 taaggtgctg gtgctgggca gcgagaacca gaataagggc aatcagacac catacgagta
     2281 tttcaatggc aaggacaact ccagggagtg gcaggagttc aaggcccgcg tggagacctc
     2341 tagatttccc aggagcaaga agcagcggat cctgctgcag aagttcgacg aggatggctt
     2401 taaggagtgc aacctgaatg acaccagata cgtgaaccgg ttcctgtgcc agtttgtggc
     2461 cgatcacatc ctgctgaccg gcaagggcaa gagaagggtg ttcgcctcta atggccagat
     2521 cacaaacctg ctgaggggat tttggggact gaggaaggtg cgggcagaga atgacagaca
     2581 ccacgcactg gatgcagtgg tggtggcatg cagcaccgtg gcaatgcagc agaagatcac
     2641 aagattcgtg aggtataagg agatgaacgc ctttgacggc aagaccatcg ataaggagac
     2701 aggcaaggtg ctgcaccaga agacccactt cccccagcct tgggagttct ttgcccagga
     2761 agtgatgatc cgggtgttcg gcaagccaga cggcaagcct gagtttgagg aggccgatac
     2821 cccagagaag ctgaggacac tgctggcaga gaagctgtct agcaggccag aggcagtgca
     2881 cgagtacgtg accccactgt tcgtgtccag ggcacccaat cggaagatgt ctggcgccca
     2941 caaggacaca ctgagaagcg ccaagaggtt tgtgaagcac aacgagaaga tctccgtgaa
     3001 gagagtgtgg ctgaccgaga tcaagctggc cgatctggag aacatggtga attacaagaa
     3061 cggcagggag atcgagctgt atgaggccct gaaggcaagg ctggaggcct acggaggaaa
     3121 tgccaagcag gccttcgacc caaaggataa ccccttttat aagaagggag gacagctggt
     3181 gaaggccgtg cgggtggaga agacccagaa gagcggcgtg ctgctgaata agaagaacgc
     3241 ctacacaatc gccgacaatg gtgatatggt gagagtggac gtgttctgta aggtggataa
     3301 gaagggcaag aatcagtact ttatcgtgcc tatctatgcc tggcaggtgg ccgagaacat
     3361 cctgccagac atcgattgca agggctacag aatcgacgat agctatacat tctgtttttc
     3421 cctgcacaag tatgacctga tcgccttcca gaaggatgag aagtccaagg tggagtttgc
     3481 ctactatatc aattgcgact cctctagcgg cgggttctac ctggcctggc acgataaggg
     3541 cagcagggag cagcggtttc gcatctccac ccagaatctg gcgctgatcc agaagtatca
     3601 ggtgaacgag ctgggcaagg agatcaggcc atgtcggctg aagaagcgcc cacccgtgcg
     3661 gtctggcggc tcaaaaagaa ccgccgacgg cagcgaattc gagcccaaga agaagaggaa
     3721 agtctaaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact
     3781 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     3841 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     3901 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     3961 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     4021 accagctggg gctcgatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     4081 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4141 ataaagtgta aagcctaggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4201 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4261 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4321 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4381 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4441 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4501 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4561 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4621 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4681 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4741 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4801 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4861 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4921 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4981 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5041 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacact
     5101 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5161 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5221 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5281 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5341 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5401 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5461 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5521 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5581 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5641 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5701 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5761 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5821 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5881 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5941 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6001 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6061 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6121 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6181 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     6241 ggagatcgat ctcccgatcc cctagggtcg actctcagta caatctgctc tgatgccgca
     6301 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
     6361 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
     6421 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
     6481 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatattgag
     6541 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     6601 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     6661 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     6721 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggccc
//
