LOCUS       sequence-412693-        7989 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of evoCjCas9 nuclease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7989)
  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 7989)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-BlastR
FEATURES             Location/Qualifiers
     source          1..7989
                     /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             3134..3154
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3164..3181
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3204..3221)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3210..3434
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3505..3521)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3505..3521)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3518..3540)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3529..3545
                     /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(3553..3559,3560..3577,3578..3583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3598..3619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    3736..3857
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3747..3766)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3801..3820
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4015..4413)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(4432..4479)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(4527..4856)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4541..4676
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4595..4614)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     4841..4861
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5240..5828)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5320..5339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5999..6790,6791..6859))
                     /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     6622..6641
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6860..6964)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7043..7062)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7412..7791
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(7792..7989,1..6)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7946..7966
                     /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 aaagtcgggt cagcccgcat cctggccttt gacatcggca tcagcagcat
      181 cggatgggcc ttcagcgaga atgacgagct gaaggactgc ggtgtgagaa tctttacaaa
      241 ggtcgagaac cccaagaccg gcgaaagcct ggcgctgcca agacggctgg cccggtctgc
      301 tcggaagaga tacgcccgga gaaaagccag actgaatcac ctgaaacacc tgatcgccaa
      361 cgagttcaaa ctgaactacg aggactacca gagcttcgat gagagcttgg ctaaggccta
      421 caaaggcagc ctgatcagcc cctacgagct gagattcaga gctctgaatg agctcctgag
      481 caagcaggac ttcgctagag tgatcctgca catcgccaag cgaagaggct acgacgacat
      541 caagaactct gatgacaagg agaagggcgc cattctgaaa gccatcaagc agaacgaaga
      601 gaaactggcc aattaccaga gcgtgggcga gtacctgtac aaggagtact tccagaagtt
      661 caaagaaaat agcaaagagt tcaccaacgt gcggaacaag aaggagtctt atgaaagatg
      721 tatcgcccag agcttcctga aagacgagct gaaactgatc ttcaagaagc aaagagagtt
      781 tggcttcagc ttcagcaaaa aatttgaaga agaggtgctg tctgtcgcct tctacaagag
      841 ggctctgaag gacttcagcc acctggtggg caattgcagc tttttcacag acgaaaagcg
      901 ggcccctaag aacagccctc tggccttcat gttcgtggct ctgaccagaa tcatcaacct
      961 gctgaacaac ctgaaaaata ccgagggcat cctctatacc aaggacgatc tgaacgccct
     1021 gctgaatgag gtgctcaaga atggcaccct gacctacaag caaacaaaaa aactgctggg
     1081 cctgtctgac gactacgagt ttaaaggcga gaagggcacc tatttcatcg aatttaagaa
     1141 gtacaaggaa ttcatcaagg cactgggcga acacaacctg tctcaggacg acctgaacga
     1201 gatcgccaag gacatcaccc tgatcaagga tgagatcaag ctgaaaaaag ctctggccaa
     1261 gtacgacctg aatcaaaacc agatcgacag cttaagcaag ctggaattta aggatcacct
     1321 gaacatctcc tttaaggccc tgaagctggt gaccccactg atgctggaag gaaagaagta
     1381 cgacgaagca tgcaacgagc ttaacctgaa ggttgctatc aacgaggata agaaggattt
     1441 cctgcctgcc tttaacgaga catactacaa ggatgaggtg accaaccccg tggtgctgag
     1501 agctatcaaa gagtacagaa aggtgctgaa cgccctgctg aagaagtacg gcaaggtcca
     1561 caagatcaat attgagctgg cccgggaggt tggaaagaac cactctcaga gagcaaagat
     1621 cgagaaggag caaaacgaga actacaaagc gaagaaggac gccgaactgg agtgcgagaa
     1681 gcttggcctg aagatcaact ctaagaatat cctgaaactc agacttttca aagaacagaa
     1741 ggaattctgt gcctacagcg gcgagaagat caaaatttct gacctgcagg atgaaaagat
     1801 gctggagatc gaccacatct acccttactc cagaagcttc gacgacagct atatgaacaa
     1861 agtgctggtg ttcacaaagc agaaccagga gaagctgaat cagacccctt tcgaggcctt
     1921 cggcaatgac tccgccaagt ggcagaaaat cgaggtgctg gccaaaaacc tgccaaccaa
     1981 gaaacagaag agaatcctcg acaagaacta caaggacaag gaacagaaga acttcaagga
     2041 tcggaacctg aacgacaccc ggtacatcgc caggctggtg ttaaattaca ccaaggacta
     2101 cctggatttc ctgcccctga gcgacgacga gaacaccaag ctgaacgaca cacagaaggg
     2161 cagcaaggtg cacgtggaag ccaagagcgg catgctgacc agcgcactgc gccacacttg
     2221 gggcttcagc gctaaggacc ggaacaacca cctgcatcac gccatcgatg ccgtgatcat
     2281 agcctacgcc aacaactcaa tcgtgaaagc tttcagtgac tttaagaaag aacaggagag
     2341 caactctgcc gaactgtacg ccaagaaaat tagcgagctg gactacaaga acaaacggaa
     2401 gttcttcgaa cctttctcag gatttagaca gaaggtgctg gataagatcg atgaaatctt
     2461 cgtgtccaag cccgagagaa agaagcctag cggagccctg cacaaggaaa ccttcagaaa
     2521 ggaagaagag ttctaccagt cttatggggg caaagaaggc gtgctgaagg ccctggaact
     2581 cggcaagatc agaaaggtga agggaaagat tgtgaagaac ggcgacatgt tcagagtgga
     2641 catcttcaag cacaagaaga ccaacaagtt ctatgccgtg cctatctaca caatggattt
     2701 cgccctgaaa gtgctgccta acaaggctgt cgccagatct aagaagggcg agatcaagga
     2761 ctggatcctg atggacgaaa actacgagtt ctgcttcagc ctgtacaagg acagcctgat
     2821 cctgatccaa acaaagaaga tgcaggagcc tgaattcgtg tactacaacg ccttcacaag
     2881 cagcaccgtg agcctgatcg tgtctaaaca tgataacaag ttcgaaaccc tgtccaagaa
     2941 ccaaaagatc ctgttcaaga acgccaacga gaaggaagtg atcgccaagg gcatcggcat
     3001 tcagaatctg aaggtgttcg agaaatatat cgtgtccgct ctgggagagg ttacaaaggc
     3061 cgagtttcgg cagagagaag attttaagaa gtctggcggc tcaaaaagaa ccgccgacgg
     3121 cagcgaattc gagcccaaga agaagaggaa agtctaaacc ggtcatcatc accatcacca
     3181 ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3241 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3301 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3361 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3421 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgatac cgtcgacctc
     3481 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     3541 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg
     3601 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3661 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3721 gcgctacaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     3781 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     3841 gctgcaataa acaagttggg gtgggcgaag aactccagca tgagatcccc gcgctggagg
     3901 atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg
     3961 gtggaatcga aatctcgtag cacgtgtcag tcctgctcct cggccacgaa gtgcttagcc
     4021 ctcccacaca taaccagagg gcagcaattc acgaatccca actgccgtcg gctgtccatc
     4081 actgtccttc actatggctt tgatcccagg atgcagatcg agaagcacct gtcggcaccg
     4141 tccgcagggg ctcaagatgc ccctgttctc atttccgatc gcgacgatac aagtcaggtt
     4201 gccagctgcc gcagcagcag cagtgcccag caccacgagt tctgcacaag gtcccccagt
     4261 aaaatgatat acattgacac cagtgaagat gcggccgtcg ctagagagag ctgcgctggc
     4321 gacgctgtag tcttcagaga tggggatgct gttgattgta gccgttgctc tttcaatgag
     4381 ggtggattct tcttgagaca aaggcttggc catggtttag ttcctcacct tgtcgtatta
     4441 tactatgccg atatactatg ccgatgatta attgtcaaca cgtgctgatc agatccgaaa
     4501 atggatatac aagctcccgg gagctttttg caaaagccta ggcctccaaa aaagcctcct
     4561 cactacttct ggaatagctc agaggcagag gcggcctcgg cctctgcata aataaaaaaa
     4621 attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc
     4681 ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg
     4741 cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt
     4801 tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc
     4861 cacagaattc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4921 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgta
     4981 agtgatctcc aaaaaataag tactttttga ctgtaaataa aattgtaagg agtaaaaagt
     5041 actttttttt ctaaaaaaat gtaattaagt aaaagtaaaa gtattgattt ttaattgtac
     5101 tcaagtaaag taaaaatccc caaaaataat acttaagtac agtaatcaag taaaattact
     5161 caagtacttt acacctctgc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5221 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5281 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5341 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5401 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5461 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5521 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5581 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5641 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5701 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5761 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5821 gatctcaaga agatcctttg atcttttcta cggggtctga cactcagtgg aacgaaaact
     5881 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5941 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6001 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6061 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6121 gtgctgcaat gataccgcgg gacccacgct caccggctcc agatttatca gcaataaacc
     6181 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6241 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6301 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6361 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6421 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6481 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6541 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6601 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6661 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6721 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6781 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6841 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6901 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6961 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
     7021 atcccctagg gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     7081 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     7141 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     7201 tgcttcgcga tgtacgggcc agatatacgc gggtaccggc atatggttct tgacagaggt
     7261 gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa gtacttaggg aaaattttac
     7321 tcaattaaaa gtaaaagtat ctggctagaa tcttacttga gtaaaagtaa aaaagtactc
     7381 cattaaaatt gtacttgagt attaacgcgt tgacattgat tattgactag ttattaatag
     7441 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     7501 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     7561 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     7621 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     7681 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     7741 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     7801 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     7861 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     7921 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     7981 tatataagc
//
