LOCUS       sequence-368925-        8207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  miniABEmax(GG)-SpCas9-NG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8207)
  AUTHORS   Xu L, Zhang C, Li H, Wang P, Gao Y, Mokadam NA, Ma J, Arnold WD, Han
            R
  TITLE     Efficient precise in vivo base editing in adult dystrophic mice.
  JOURNAL   Nat Commun. 2021 Jun 17;12(1):3719. doi: 10.1038/s41467-021-23996-y.
  PUBMED    34140489
REFERENCE   2  (bases 1 to 8207)
  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 
            Commun."; date: "2021-06-17"; volume: "12(1):3719. doi"; pages: " 
            10.1038/s41467-021-23996-y"
COMMENT     Sequence Label: pCMV_miniABEmaxNG(GG)
FEATURES             Location/Qualifiers
     source          1..8207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        154..357
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     308..328
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      400..409
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             451..471
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1072..5172
                     /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
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5215..5235
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5244..5261
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5284..5301)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5290..5514
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5585..5601)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5585..5601)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5598..5620)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5609..5625
                     /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(5633..5639,5640..5657,5658..5663))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5678..5699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5816..5833)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5987..6575)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6067..6086)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6746..7537,7538..7606))
                     /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     7369..7388
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7607..7711)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7790..7809)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7981..8207,1..153)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
       61 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      121 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      181 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      241 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      301 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
      361 ttagtgaacc gtcagatccg ctagagatcc gcggccgccg ccaccatggc tagcatgaaa
      421 cggacagccg acggaagcga gttcgagtca ccaaagaaga agcggaaagt cggatcctct
      481 gaggtggagt tttcccacga gtactggatg agacatgccc tgaccctggc caagagggca
      541 cgcgatgaga gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag agtgatcggc
      601 gagggctgga acagagccat cggcctgcac gacccaacag gccatgccga aattatggcc
      661 ctgaggcagg gcggcctggt catgcagaac tacagactga ttgacgccac cctgtacggg
      721 acattcgagc cttgcgtgat gtgcgccggc gccatgatcc actctaggat cggccgcgtg
      781 gtgtttggcg tgaggaacgc aaaaaccggc gccgcaggct ccctgatgga cgtgctgcac
      841 taccccggca tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc
      901 gccctgctgt gctatttctt tcggatgcct agacaggtgt tcaatgctca gaagaaggcc
      961 cagagctcca ccgactccgg aggatctagc ggaggctcct ctggctctga gacacctggc
     1021 acaagcgaga gcgcaacacc tgaaagcagc gggggcagca gcggggggtc agacaagaag
     1081 tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     1141 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     1201 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     1261 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     1321 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     1381 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     1441 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     1501 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     1561 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     1621 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     1681 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     1741 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     1801 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     1861 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     1921 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     1981 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     2041 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     2101 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     2161 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     2221 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     2281 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     2341 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     2401 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     2461 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     2521 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     2581 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     2641 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     2701 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     2761 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     2821 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     2881 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     2941 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     3001 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     3061 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     3121 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     3181 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     3241 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     3301 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     3361 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     3421 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     3481 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     3541 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     3601 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     3661 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     3721 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     3781 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     3841 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     3901 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     3961 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     4021 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     4081 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     4141 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     4201 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     4261 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     4321 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     4381 ttcagcaaag agtctatcag gcccaagagg aacagcgata agctgatcgc cagaaagaag
     4441 gactgggacc ctaagaagta cggcggcttc gtcagcccca ccgtggccta ttctgtgctg
     4501 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     4561 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     4621 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     4681 gagctggaaa acggccggaa gagaatgctg gcctctgcca gattcctgca gaagggaaac
     4741 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     4801 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     4861 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     4921 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     4981 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tagggccttc
     5041 aagtactttg acaccaccat cgaccggaag gtgtacagga gcaccaaaga ggtgctggac
     5101 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     5161 ctgggaggtg actctggcgg ctcaaaaaga accgccgacg gcagcgaatt cgagcccaag
     5221 aagaagagga aagtctaacc ggtcatcatc accatcacca ttgagtttaa acccgctgat
     5281 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     5341 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     5401 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     5461 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     5521 aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc ttggcgtaat
     5581 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5641 gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa ctcacattaa
     5701 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5761 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5821 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5881 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5941 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     6001 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6061 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6121 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6181 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6241 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6301 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6361 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6421 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6481 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6541 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6601 ggtctgacac tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6661 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6721 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6781 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6841 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6901 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6961 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     7021 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     7081 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7141 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7201 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7261 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7321 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7381 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7441 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7501 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7561 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7621 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7681 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7741 tcgacggatc gggagatcga tctcccgatc ccctagggtc gactctcagt acaatctgct
     7801 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     7861 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     7921 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     7981 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     8041 catatattga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     8101 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     8161 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccac
//
