LOCUS       sequence-265158-        6324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the N-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 6324)
  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 6324)
  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 N-terminal
FEATURES             Location/Qualifiers
     source          1..6324
                     /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             466..1149
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             1246..2961
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             3310..3330
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3339..3356
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3379..3396)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3385..3609
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3680..3696)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3680..3696)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3693..3715)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3704..3720
                     /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(3728..3734,3735..3752,3753..3758))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3773..3794
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3911..3928)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4082..4670)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4162..4181)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4841..5632,5633..5701))
                     /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     5464..5483
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5702..5806)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5885..5904)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6076..6324,1..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 aagtctcctc agagactggg
      481 cctgtcgccg tcgatccaac cctgcgccgc cggattgaac ctcacgagtt tgaagtgttc
      541 tttgaccccc gggagctgag aaaggagaca tgcctgctgt acgagatcaa ctggggaggc
      601 aggcactcca tctggaggca cacctctcag aacacaaata agcacgtgga ggtgaacttc
      661 atcgagaagt ttaccacaga gcggtacttc tgccccaata ccagatgtag catcacatgg
      721 tttctgagct ggtccccttg cggagagtgt agcagggcca tcaccgagtt cctgtccaga
      781 tatccacacg tgacactgtt tatctacatc gccaggctgt atcaccacgc agacccaagg
      841 aataggcagg gcctgcgcga tctgatcagc tccggcgtga ccatccagat catgacagag
      901 caggagtccg gctactgctg gcggaacttc gtgaattatt ctcctagcaa cgaggcccac
      961 tggcctaggt acccacacct gtgggtgcgc ctgtacgtgc tggagctgta ttgcatcatc
     1021 ctgggcctgc ccccttgtct gaatatcctg cggagaaagc agccccagct gaccttcttt
     1081 acaatcgccc tgcagtcttg tcactatcag aggctgccac cccacatcct gtgggccaca
     1141 ggcctgaagt ctggaggatc tagcggagga tcctctggca gcgagacacc aggaacaagc
     1201 gagtcagcaa caccagagag cagtggcggc agcagcggcg gcagcgacaa gaagtacagc
     1261 atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag
     1321 gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac
     1381 ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga
     1441 accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc
     1501 agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg
     1561 gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg
     1621 gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc
     1681 gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc
     1741 cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc
     1801 cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg
     1861 gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc
     1921 gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg
     1981 ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg
     2041 agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac
     2101 gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg
     2161 agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac
     2221 gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag
     2281 tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga
     2341 gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc
     2401 gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac
     2461 aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag
     2521 gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc
     2581 cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc
     2641 agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct
     2701 tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag
     2761 gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa
     2821 gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag
     2881 gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag
     2941 gactacttca agaaaatcga gtgcctgtcc tacgagacag agatcctgac agtggagtat
     3001 ggcctgctgc caatcggcaa gatcgtggag aagaggatcg agtgtaccgt gtactctgtg
     3061 gataacaatg gcaacatcta tacacagccc gtggcacagt ggcacgatag gggagagcag
     3121 gaggtgttcg agtattgcct ggaggacggc agcctgatca gggcaaccaa ggaccacaag
     3181 ttcatgacag tggatggcca gatgctgccc atcgacgaga ttttcgagcg ggagctggac
     3241 ctgatgagag tggataacct gcctaatagc ggaggcagta aaagaacagc agacgggagt
     3301 gagtttgagc ccaagaaaaa gagaaaggtg taaccggtca tcatcaccat caccattgag
     3361 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     3421 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3481 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3541 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3601 gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg acctctagct
     3661 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3721 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga
     3781 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3841 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3901 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3961 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4021 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4081 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4141 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4201 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4261 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4321 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4381 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4441 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4501 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4561 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4621 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4681 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4741 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4801 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4861 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4921 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4981 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5041 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5101 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5161 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5221 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5281 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5341 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5401 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5461 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5521 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5581 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5641 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5701 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5761 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5821 aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc
     5881 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     5941 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     6001 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     6061 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     6121 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     6181 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     6241 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     6301 acttggcagt acatcaagtg tatc
//
