LOCUS       sequence-373130-        8217 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpRY-HF1-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-HF1-ABE8e
FEATURES             Location/Qualifiers
     source          1..8217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        454..657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     608..628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     699..718
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        699..717
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             767..787
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1382..5482
                     /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
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5525..5545
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5554..5571
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5594..5611)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5600..5824
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5895..5911)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5895..5911)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5908..5930)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5919..5935
                     /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(5943..5949,5950..5967,5968..5973))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5988..6009
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6126..6143)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6297..6885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6377..6396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7056..7847,7848..7916))
                     /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     7679..7698
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7917..8021)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8100..8119)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
       61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat tgagttccgc gttacataac ttacggtaaa tggcccgcct
      181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
      661 ttagtgaacc gtcagatccg ctagagatcc gcggccgcta atacgactca ctatagggag
      721 agccgccacc atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg
      781 gaaagtctct gaggtggagt tttcccacga gtactggatg agacatgccc tgaccctggc
      841 caagagggca cgggatgaga gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag
      901 agtgatcggc gagggctgga acagagccat cggcctgcac gacccaacag cccatgccga
      961 aattatggcc ctgagacagg gcggcctggt catgcagaac tacagactga ttgacgccac
     1021 cctgtacgtg acattcgagc cttgcgtgat gtgcgccggc gccatgatcc actctaggat
     1081 cggccgcgtg gtgtttggcg tgaggaactc aaaaagaggc gccgcaggct ccctgatgaa
     1141 cgtgctgaac taccccggca tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga
     1201 tgaatgtgcc gccctgctgt gcgatttcta tcggatgcct agacaggtgt tcaatgctca
     1261 gaagaaggcc cagagctcca tcaactctgg aggatctagc ggaggatcct ctggcagcga
     1321 gacaccagga acaagcgagt cagcaacacc agagagcagt ggcggcagca gcggcggcag
     1381 cgacaagaag tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat
     1441 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca
     1501 cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgagag
     1561 aacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
     1621 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact
     1681 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
     1741 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
     1801 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat
     1861 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt
     1921 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat
     1981 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
     2041 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct
     2101 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga
     2161 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca
     2221 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct
     2281 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
     2341 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
     2401 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg
     2461 ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga
     2521 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa
     2581 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
     2641 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
     2701 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag
     2761 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt
     2821 ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccgcct tcgataagaa
     2881 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
     2941 taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
     3001 cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt
     3061 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc
     3121 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat
     3181 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
     3241 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
     3301 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcgc
     3361 cctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga
     3421 tttcctgaag tccgacggct tcgccaacag aaacttcatg gccctgatcc acgacgacag
     3481 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
     3541 cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt
     3601 gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat
     3661 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat
     3721 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt
     3781 ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga
     3841 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat
     3901 cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga
     3961 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa
     4021 ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac
     4081 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
     4141 ggtggaaacc cgggccatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
     4201 taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa
     4261 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta
     4321 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta
     4381 ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
     4441 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
     4501 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc
     4561 tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc
     4621 caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca
     4681 gacaggcggc ttcagcaaag agtctatcag acccaagagg aacagcgata agctgatcgc
     4741 cagaaagaag gactgggacc ctaagaagta cggcggcttc ctgtggccca ccgtggccta
     4801 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa
     4861 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt
     4921 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta
     4981 ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgcca agcagctgca
     5041 gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca
     5101 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca
     5161 gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat
     5221 cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc
     5281 catcagagag caggccgaga atatcatcca cctgtttacc ctgaccagac tgggagcccc
     5341 tagagccttc aagtactttg acaccaccat cgaccccaag cagtacagaa gcaccaaaga
     5401 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga
     5461 cctgtctcag ctgggaggtg actctggcgg ctcaaaaaga accgccgacg gcagcgaatt
     5521 cgagcccaag aagaagagga aagtctaacc ggtcatcatc accatcacca ttgagtttaa
     5581 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     5641 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     5701 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     5761 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     5821 atggcttctg aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc
     5881 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5941 cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa
     6001 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6061 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     6121 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     6181 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     6241 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6301 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     6361 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6421 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6481 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6541 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6601 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6661 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6721 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6781 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6841 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6901 ttttctacgg ggtctgacac tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6961 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     7021 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     7081 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     7141 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     7201 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     7261 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7321 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     7381 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7441 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7501 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7561 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7621 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     7681 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7741 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7801 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7861 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7921 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7981 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     8041 ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc gactctcagt
     8101 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     8161 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgac
//
