LOCUS       sequence-409518-        8217 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpRY-ABE8e in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8217)
  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 8217)
  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-SpRY-ABE8e
FEATURES             Location/Qualifiers
     source          1..8217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        135..514
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..718
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     669..689
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     760..779
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        760..778
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             828..848
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1443..5543
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5586..5606
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5615..5632
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5655..5672)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5661..5885
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5956..5972)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5956..5972)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5969..5991)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5980..5996
                     /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(6004..6010,6011..6028,6029..6034))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6049..6070
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6187..6204)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6358..6946)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6438..6457)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7117..7908,7909..7977))
                     /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     7740..7759
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7978..8082)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8161..8180)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
       61 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
      121 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      181 attagttcat agcccatata ttgagttccg cgttacataa cttacggtaa atggcccgcc
      241 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      301 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      361 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      421 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      481 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      541 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      601 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      661 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
      721 tttagtgaac cgtcagatcc gctagagatc cgcggccgct aatacgactc actataggga
      781 gagccgccac catgaaacgg acagccgacg gaagcgagtt cgagtcacca aagaagaagc
      841 ggaaagtctc tgaggtggag ttttcccacg agtactggat gagacatgcc ctgaccctgg
      901 ccaagagggc acgggatgag agggaggtgc ctgtgggagc cgtgctggtg ctgaacaata
      961 gagtgatcgg cgagggctgg aacagagcca tcggcctgca cgacccaaca gcccatgccg
     1021 aaattatggc cctgagacag ggcggcctgg tcatgcagaa ctacagactg attgacgcca
     1081 ccctgtacgt gacattcgag ccttgcgtga tgtgcgccgg cgccatgatc cactctagga
     1141 tcggccgcgt ggtgtttggc gtgaggaact caaaaagagg cgccgcaggc tccctgatga
     1201 acgtgctgaa ctaccccggc atgaatcacc gcgtcgaaat taccgaggga atcctggcag
     1261 atgaatgtgc cgccctgctg tgcgatttct atcggatgcc tagacaggtg ttcaatgctc
     1321 agaagaaggc ccagagctcc atcaactctg gaggatctag cggaggatcc tctggcagcg
     1381 agacaccagg aacaagcgag tcagcaacac cagagagcag tggcggcagc agcggcggca
     1441 gcgacaagaa gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga
     1501 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc
     1561 acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgaga
     1621 gaacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct
     1681 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac
     1741 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca
     1801 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga
     1861 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca
     1921 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg
     1981 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca
     2041 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac
     2101 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc
     2161 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg
     2221 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc
     2281 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc
     2341 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta
     2401 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc
     2461 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg
     2521 gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg
     2581 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga
     2641 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg
     2701 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg
     2761 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca
     2821 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag
     2881 tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga
     2941 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt
     3001 ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga
     3061 gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg
     3121 tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct
     3181 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta
     3241 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc
     3301 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc
     3361 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca
     3421 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg
     3481 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca
     3541 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc
     3601 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag
     3661 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga
     3721 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa
     3781 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg
     3841 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg
     3901 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata
     3961 tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg
     4021 acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga
     4081 actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga
     4141 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc
     4201 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca
     4261 ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca
     4321 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact
     4381 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt
     4441 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga
     4501 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca
     4561 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc
     4621 ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg
     4681 ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc
     4741 agacaggcgg cttcagcaaa gagtctatca gacccaagag gaacagcgat aagctgatcg
     4801 ccagaaagaa ggactgggac cctaagaagt acggcggctt cctgtggccc accgtggcct
     4861 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga
     4921 aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact
     4981 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt
     5041 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc aagcagctgc
     5101 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
     5161 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
     5221 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
     5281 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
     5341 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaga ctgggagccc
     5401 ctagagcctt caagtacttt gacaccacca tcgaccccaa gcagtacaga agcaccaaag
     5461 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
     5521 acctgtctca gctgggaggt gactctggcg gctcaaaaag aaccgccgac ggcagcgaat
     5581 tcgagcccaa gaagaagagg aaagtctaac cggtcatcat caccatcacc attgagttta
     5641 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     5701 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     5761 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     5821 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     5881 tatggcttct gaggcggaaa gaaccagctg gggctcgata ccgtcgacct ctagctagag
     5941 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     6001 acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat gagtgagcta
     6061 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6121 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     6181 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     6241 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     6301 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6361 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6421 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6481 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6541 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6601 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6661 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6721 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6781 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6841 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6901 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6961 cttttctacg gggtctgaca ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     7021 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     7081 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     7141 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7201 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     7261 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     7321 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     7381 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     7441 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7501 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7561 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7621 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7681 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7741 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7801 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7861 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7921 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7981 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     8041 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     8101 gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt cgactctcag
     8161 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgtt
//
