LOCUS       sequence-270140-        6291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the C-terminal split-intein fragment of BE3.9max from the 
            CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6291)
  AUTHORS   Levy JM, Yeh WH, Pendse N, Davis JR, Hennessey E, Butcher R, Koblan 
            LW, Comander J, Liu Q, Liu DR
  TITLE     Cytosine and adenine base editing of the brain, liver, retina, heart
            and skeletal muscle of mice via adeno-associated viruses.
  JOURNAL   Nat Biomed Eng. 2020 Jan;4(1):97-110. doi: 
            10.1038/s41551-019-0501-5. Epub 2020 Jan 14.
  PUBMED    31937940
REFERENCE   2  (bases 1 to 6291)
  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"; doi: 
            "10.1038/s41551-019-0501-5"; journalName: "Nat Biomed Eng"; date: 
            "2020-01"; volume: "4"; issue: "1"; pages: "97-110"
COMMENT     Sequence Label: CMV_Npu-BE3.9max C-terminal
FEATURES             Location/Qualifiers
     source          1..6291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             571..2955
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(C)
                     /translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
                     IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
                     TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
                     ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
                     LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
                     KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
                     AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
                     QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
                     FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             2986..3234
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3277..3297
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3306..3323
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3346..3363)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3352..3576
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3647..3663)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3647..3663)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3660..3682)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3671..3687
                     /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(3695..3701,3702..3719,3720..3725))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3740..3761
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3878..3895)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4049..4637)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4129..4148)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4808..5599,5600..5668))
                     /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     5431..5450
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5669..5773)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5852..5871)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6043..6291,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcatcaa gattgctaca
      481 cggaaatacc tgggaaagca gaacgtgtac gacatcggcg tggagcggga tcacaacttc
      541 gccctgaaga atggctttat cgccagcaat tgcttcgact ccgtggaaat ctccggcgtg
      601 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
      661 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
      721 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
      781 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
      841 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
      901 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
      961 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
     1021 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
     1081 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
     1141 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
     1201 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac
     1261 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
     1321 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct
     1381 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac
     1441 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
     1501 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc
     1561 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
     1621 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
     1681 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
     1741 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
     1801 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
     1861 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
     1921 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
     1981 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc
     2041 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg
     2101 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc
     2161 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag
     2221 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg
     2281 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg
     2341 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa
     2401 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg
     2461 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga
     2521 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag
     2581 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag
     2641 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc
     2701 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga
     2761 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc
     2821 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg
     2881 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct
     2941 cagctgggag gtgacagcgg cgggagcggc gggagcgggg ggagcactaa tctgagcgac
     3001 atcattgaga aggagactgg gaaacagctg gtcattcagg agtccatcct gatgctgcct
     3061 gaggaggtgg aggaagtgat cggcaacaag ccagagtctg acatcctggt gcacaccgcc
     3121 tacgacgagt ccacagatga gaatgtgatg ctgctgacct ctgacgcccc cgagtataag
     3181 ccttgggccc tggtcatcca ggattctaac ggcgagaata agatcaagat gctgagcgga
     3241 ggatccaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctaa
     3301 ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt
     3361 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3421 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3481 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3541 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3601 ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     3661 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3721 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3781 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3841 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3901 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3961 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4021 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4081 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4141 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4201 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4261 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4321 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4381 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4441 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4501 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4561 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4621 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac actcagtgga
     4681 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4741 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4801 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4861 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4921 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4981 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5041 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5101 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5161 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5221 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5281 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5341 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5401 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5461 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5521 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5581 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5641 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5701 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5761 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     5821 gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa
     5881 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     5941 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     6001 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     6061 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     6121 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     6181 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     6241 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat c
//
