LOCUS       sequence-412694-        8577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of evoCjCas9 ABE8e.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8577)
  AUTHORS   Schmidheini L, Mathis N, Marquart KF, Rothgangl T, Kissling L, Bock 
            D, Chanez C, Wang JP, Jinek M, Schwank G
  TITLE     Continuous directed evolution of a compact CjCas9 variant with broad
            PAM compatibility.
  JOURNAL   Nat Chem Biol. 2023 Sep 21. doi: 10.1038/s41589-023-01427-x.
  PUBMED    37735239
REFERENCE   2  (bases 1 to 8577)
  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 Chem 
            Biol. 2023 Sep 21. doi: 10.1038/s41589-023-01427-x."
COMMENT     Sequence Label: p2T-CMV-evoCjCas9-ABE8e-BlastR
FEATURES             Location/Qualifiers
     source          1..8577
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     48..67
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        48..66
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             116..136
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3722..3742
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3752..3769
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3792..3809)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3798..4022
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4093..4109)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4093..4109)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4106..4128)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4117..4133
                     /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(4141..4147,4148..4165,4166..4171))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4186..4207
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    4324..4445
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4335..4354)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4389..4408
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4603..5001)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(5020..5067)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5115..5444)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5129..5264
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5183..5202)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5429..5449
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     misc_feature    join(5568..5755,5756..5767)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     rep_origin      complement(5828..6416)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5908..5927)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6587..7378,7379..7447))
                     /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     7210..7229
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7448..7552)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7631..7650)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    join(7842..7853,7854..7991)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     enhancer        8000..8379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8380..8577,1..6)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8534..8554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 agagctggtt tagtgaaccg tcagatccgc tagagatccg cggccgctaa tacgactcac
       61 tatagggaga gccgccacca tgaaacggac agccgacgga agcgagttcg agtcaccaaa
      121 gaagaagcgg aaagtctctg aggtggagtt ttcccacgag tactggatga gacatgccct
      181 gaccctggcc aagagggcac gggatgagag ggaggtgcct gtgggagccg tgctggtgct
      241 gaacaataga gtgatcggcg agggctggaa cagagccatc ggcctgcacg acccaacagc
      301 ccatgccgaa attatggccc tgagacaggg cggcctggtc atgcagaact acagactgat
      361 tgacgccacc ctgtacgtga cattcgagcc ttgcgtgatg tgcgccggcg ccatgatcca
      421 ctctaggatc ggccgcgtgg tgtttggcgt gaggaactca aaaagaggcg ccgcaggctc
      481 cctgatgaac gtgctgaact accccggcat gaatcaccgc gtcgaaatta ccgagggaat
      541 cctggcagat gaatgtgccg ccctgctgtg cgatttctat cggatgccta gacaggtgtt
      601 caatgctcag aagaaggccc agagctccat caactccgga ggatctagcg gaggctcctc
      661 tggctctgag acacctggca caagcgagag cgcaacacct gaaagcagcg ggggcagcag
      721 cggggggtca gcccgcatcc tggcctttgc catcggcatc agcagcatcg gatgggcctt
      781 cagcgagaat gacgagctga aggactgcgg tgtgagaatc tttacaaagg tcgagaaccc
      841 caagaccggc gaaagcctgg cgctgccaag acggctggcc cggtctgctc ggaagagata
      901 cgcccggaga aaagccagac tgaatcacct gaaacacctg atcgccaacg agttcaaact
      961 gaactacgag gactaccaga gcttcgatga gagcttggct aaggcctaca aaggcagcct
     1021 gatcagcccc tacgagctga gattcagagc tctgaatgag ctcctgagca agcaggactt
     1081 cgctagagtg atcctgcaca tcgccaagcg aagaggctac gacgacatca agaactctga
     1141 tgacaaggag aagggcgcca ttctgaaagc catcaagcag aacgaagaga aactggccaa
     1201 ttaccagagc gtgggcgagt acctgtacaa ggagtacttc cagaagttca aagaaaatag
     1261 caaagagttc accaacgtgc ggaacaagaa ggagtcttat gaaagatgta tcgcccagag
     1321 cttcctgaaa gacgagctga aactgatctt caagaagcaa agagagtttg gcttcagctt
     1381 cagcaaaaaa tttgaagaag aggtgctgtc tgtcgccttc tacaagaggg ctctgaagga
     1441 cttcagccac ctggtgggca attgcagctt tttcacagac gaaaagcggg cccctaagaa
     1501 cagccctctg gccttcatgt tcgtggctct gaccagaatc atcaacctgc tgaacaacct
     1561 gaaaaatacc gagggcatcc tctataccaa ggacgatctg aacgccctgc tgaatgaggt
     1621 gctcaagaat ggcaccctga cctacaagca aacaaaaaaa ctgctgggcc tgtctgacga
     1681 ctacgagttt aaaggcgaga agggcaccta tttcatcgaa tttaagaagt acaaggaatt
     1741 catcaaggca ctgggcgaac acaacctgtc tcaggacgac ctgaacgaga tcgccaagga
     1801 catcaccctg atcaaggatg agatcaagct gaaaaaagct ctggccaagt acgacctgaa
     1861 tcaaaaccag atcgacagct taagcaagct ggaatttaag gatcacctga acatctcctt
     1921 taaggccctg aagctggtga ccccactgat gctggaagga aagaagtacg acgaagcatg
     1981 caacgagctt aacctgaagg ttgctatcaa cgaggataag aaggatttcc tgcctgcctt
     2041 taacgagaca tactacaagg atgaggtgac caaccccgtg gtgctgagag ctatcaaaga
     2101 gtacagaaag gtgctgaacg ccctgctgaa gaagtacggc aaggtccaca agatcaatat
     2161 tgagctggcc cgggaggttg gaaagaacca ctctcagaga gcaaagatcg agaaggagca
     2221 aaacgagaac tacaaagcga agaaggacgc cgaactggag tgcgagaagc ttggcctgaa
     2281 gatcaactct aagaatatcc tgaaactcag acttttcaaa gaacagaagg aattctgtgc
     2341 ctacagcggc gagaagatca aaatttctga cctgcaggat gaaaagatgc tggagatcga
     2401 ccacatctac ccttactcca gaagcttcga cgacagctat atgaacaaag tgctggtgtt
     2461 cacaaagcag aaccaggaga agctgaatca gacccctttc gaggccttcg gcaatgactc
     2521 cgccaagtgg cagaaaatcg aggtgctggc caaaaacctg ccaaccaaga aacagaagag
     2581 aatcctcgac aagaactaca aggacaagga acagaagaac ttcaaggatc ggaacctgaa
     2641 cgacacccgg tacatcgcca ggctggtgtt aaattacacc aaggactacc tggatttcct
     2701 gcccctgagc gacgacgaga acaccaagct gaacgacaca cagaagggca gcaaggtgca
     2761 cgtggaagcc aagagcggca tgctgaccag cgcactgcgc cacacttggg gcttcagcgc
     2821 taaggaccgg aacaaccacc tgcatcacgc catcgatgcc gtgatcatag cctacgccaa
     2881 caactcaatc gtgaaagctt tcagtgactt taagaaagaa caggagagca actctgccga
     2941 actgtacgcc aagaaaatta gcgagctgga ctacaagaac aaacggaagt tcttcgaacc
     3001 tttctcagga tttagacaga aggtgctgga taagatcgat gaaatcttcg tgtccaagcc
     3061 cgagagaaag aagcctagcg gagccctgca caaggaaacc ttcagaaagg aagaagagtt
     3121 ctaccagtct tatgggggca aagaaggcgt gctgaaggcc ctggaactcg gcaagatcag
     3181 aaaggtgaag ggaaagattg tgaagaacgg cgacatgttc agagtggaca tcttcaagca
     3241 caagaagacc aacaagttct atgccgtgcc tatctacaca atggatttcg ccctgaaagt
     3301 gctgcctaac aaggctgtcg ccagatctaa gaagggcgag atcaaggact ggatcctgat
     3361 ggacgaaaac tacgagttct gcttcagcct gtacaaggac agcctgatcc tgatccaaac
     3421 aaagaagatg caggagcctg aattcgtgta ctacaacgcc ttcacaagca gcaccgtgag
     3481 cctgatcgtg tctaaacatg ataacaagtt cgaaaccctg tccaagaacc aaaagatcct
     3541 gttcaagaac gccaacgaga aggaagtgat cgccaagggc atcggcattc agaatctgaa
     3601 ggtgttcgag aaatatatcg tgtccgctct gggagaggtt acaaaggccg agtttcggca
     3661 gagagaagat tttaagaagt ctggcggctc aaaaagaacc gccgacggca gcgaattcga
     3721 gcccaagaag aagaggaaag tctaaaccgg tcatcatcac catcaccatt gagtttaaac
     3781 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3841 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3901 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3961 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     4021 ggcttctgag gcggaaagaa ccagctgggg ctcgataccg tcgacctcta gctagagctt
     4081 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4141 caacatacga gccggaagca taaagtgtaa agcctagggt gcctaatgag tgagctaact
     4201 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4261 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctacagaca
     4321 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     4381 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     4441 aagttggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagccg
     4501 gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt ggaatcgaaa
     4561 tctcgtagca cgtgtcagtc ctgctcctcg gccacgaagt gcttagccct cccacacata
     4621 accagagggc agcaattcac gaatcccaac tgccgtcggc tgtccatcac tgtccttcac
     4681 tatggctttg atcccaggat gcagatcgag aagcacctgt cggcaccgtc cgcaggggct
     4741 caagatgccc ctgttctcat ttccgatcgc gacgatacaa gtcaggttgc cagctgccgc
     4801 agcagcagca gtgcccagca ccacgagttc tgcacaaggt cccccagtaa aatgatatac
     4861 attgacacca gtgaagatgc ggccgtcgct agagagagct gcgctggcga cgctgtagtc
     4921 ttcagagatg gggatgctgt tgattgtagc cgttgctctt tcaatgaggg tggattcttc
     4981 ttgagacaaa ggcttggcca tggtttagtt cctcaccttg tcgtattata ctatgccgat
     5041 atactatgcc gatgattaat tgtcaacacg tgctgatcag atccgaaaat ggatatacaa
     5101 gctcccggga gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg
     5161 aatagctcag aggcagaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca
     5221 tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc
     5281 gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     5341 cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg
     5401 cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca cagaattcac
     5461 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5521 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtaag tgatctccaa
     5581 aaaataagta ctttttgact gtaaataaaa ttgtaaggag taaaaagtac ttttttttct
     5641 aaaaaaatgt aattaagtaa aagtaaaagt attgattttt aattgtactc aagtaaagta
     5701 aaaatcccca aaaataatac ttaagtacag taatcaagta aaattactca agtactttac
     5761 acctctgcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5821 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5881 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5941 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6001 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6061 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6121 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6181 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6241 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6301 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6361 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6421 atcctttgat cttttctacg gggtctgaca ctcagtggaa cgaaaactca cgttaaggga
     6481 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6541 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6601 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6661 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6721 taccgcggga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6781 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6841 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6901 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6961 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7021 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7081 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7141 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7201 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7261 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7321 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7381 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7441 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7501 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7561 cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt
     7621 cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     7681 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     7741 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     7801 tacgggccag atatacgcgg gtaccggcat atggttcttg acagaggtgt aaaaagtact
     7861 caaaaatttt actcaagtga aagtacaagt acttagggaa aattttactc aattaaaagt
     7921 aaaagtatct ggctagaatc ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt
     7981 acttgagtat taacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     8041 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     8101 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     8161 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     8221 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     8281 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     8341 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     8401 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     8461 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     8521 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagc
//
