LOCUS       sequence-369024-        8841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ABEmax-SpCas9-NG-loop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8841)
  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 8841)
  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_ABE‐NG‐loop
FEATURES             Location/Qualifiers
     source          1..8841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(534..1325,1326..1394))
                     /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     1157..1176
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1395..1499)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1578..1597)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1769..2148
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2149..2352
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2303..2323
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2394..2413
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2394..2412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2462..2482
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5303..7801
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(C)
                     /translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI
                     SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY
                     AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH
                     DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE
                     NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL
                     QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV
                     VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI
                     LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV
                     GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL
                     ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIR
                     PKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME
                     RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNELALP
                     SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD
                     KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATL
                     IHQSITGLYETRIDLSQLGGD"
     CDS             7844..7864
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7873..7890
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7913..7930)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7919..8143
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(8214..8230)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8214..8230)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8227..8249)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8238..8254
                     /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(8262..8268,8269..8286,8287..8292))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8307..8328
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8445..8462)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(8616..8841,1..363))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8696..8715)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
       61 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
      121 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
      181 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
      241 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
      301 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
      361 caagaagatc ctttgatctt ttctacgggg tctgacactc agtggaacga aaactcacgt
      421 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      481 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      541 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
      601 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
      661 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
      721 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
      781 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
      841 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      901 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      961 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     1021 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     1081 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     1141 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     1201 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1261 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1321 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1381 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1441 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1501 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc tcccgatccc
     1561 ctagggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     1621 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     1681 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     1741 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
     1801 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     1861 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     1921 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     1981 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     2041 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     2101 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     2161 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     2221 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     2281 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     2341 ataagcagag ctggtttagt gaaccgtcag atccgctaga gatccgcggc cgctaatacg
     2401 actcactata gggagagccg ccaccatgaa acggacagcc gacggaagcg agttcgagtc
     2461 accaaagaag aagcggaaag tctctgaagt cgagtttagc cacgagtatt ggatgaggca
     2521 cgcactgacc ctggcaaagc gagcatggga tgaaagagaa gtccccgtgg gcgccgtgct
     2581 ggtgcacaac aatagagtga tcggagaggg atggaacagg ccaatcggcc gccacgaccc
     2641 taccgcacac gcagagatca tggcactgag gcagggaggc ctggtcatgc agaattaccg
     2701 cctgatcgat gccaccctgt atgtgacact ggagccatgc gtgatgtgcg caggagcaat
     2761 gatccacagc aggatcggaa gagtggtgtt cggagcacgg gacgccaaga ccggcgcagc
     2821 aggctccctg atggatgtgc tgcaccaccc cggcatgaac caccgggtgg agatcacaga
     2881 gggaatcctg gcagacgagt gcgccgccct gctgagcgat ttctttagaa tgcggagaca
     2941 ggagatcaag gcccagaaga aggcacagag ctccaccgac tctggaggat ctagcggagg
     3001 atcctctgga agcgagacac caggcacaag cgagtccgcc acaccagaga gctccggcgg
     3061 ctcctccgga ggatcctctg aggtggagtt ttcccacgag tactggatga gacatgccct
     3121 gaccctggcc aagagggcac gcgatgagag ggaggtgcct gtgggagccg tgctggtgct
     3181 gaacaataga gtgatcggcg agggctggaa cagagccatc ggcctgcacg acccaacagc
     3241 ccatgccgaa attatggccc tgagacaggg cggcctggtc atgcagaact acagactgat
     3301 tgacgccacc ctgtacgtga cattcgagcc ttgcgtgatg tgcgccggcg ccatgatcca
     3361 ctctaggatc ggccgcgtgg tgtttggcgt gaggaacgca aaaaccggcg ccgcaggctc
     3421 cctgatggac gtgctgcact accccggcat gaatcaccgc gtcgaaatta ccgagggaat
     3481 cctggcagat gaatgtgccg ccctgctgtg ctatttcttt cggatgccta gacaggtgtt
     3541 caatgctcag aagaaggccc agagctccac cgactccgga ggatctagcg gaggctcctc
     3601 tggctctgag acacctggca caagcgagag cgcaacacct gaaagcagcg ggggcagcag
     3661 cggggggtca gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg
     3721 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     3781 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
     3841 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     3901 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     3961 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     4021 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     4081 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     4141 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     4201 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     4261 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     4321 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     4381 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     4441 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     4501 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     4561 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     4621 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     4681 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     4741 ctacgccggc tacgtgggcg ccgacaagaa gctgcgcaag cgcagctcta aactggccac
     4801 agaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     4861 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     4921 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     4981 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     5041 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     5101 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     5161 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     5221 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     5281 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     5341 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     5401 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     5461 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     5521 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     5581 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     5641 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     5701 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     5761 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     5821 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     5881 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     5941 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     6001 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     6061 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     6121 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     6181 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
     6241 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     6301 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     6361 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     6421 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     6481 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     6541 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     6601 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     6661 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
     6721 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     6781 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     6841 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
     6901 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     6961 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     7021 gtctatccgg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     7081 taagaagtac ggcggcttcg tcagccccac cgtggcctat tctgtgctgg tggtggccaa
     7141 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     7201 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     7261 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     7321 cggccggaag agaatgctgg cctctgcccg ctttctgcag aagggaaacg aactggccct
     7381 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     7441 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     7501 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     7561 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     7621 tatcatccac ctgtttaccc tgaccaatct gggagcccct cgggccttca agtactttga
     7681 caccaccatc gaccggaagg tgtaccggag caccaaagag gtgctggacg ccaccctgat
     7741 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggtga
     7801 ctctggcggc tcaaaaagaa ccgccgacgg cagcgaattc gagcccaaga agaagaggaa
     7861 agtctaaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact
     7921 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     7981 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     8041 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     8101 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     8161 accagctggg gctcgatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     8221 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     8281 ataaagtgta aagcctaggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     8341 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     8401 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     8461 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     8521 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     8581 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     8641 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     8701 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     8761 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     8821 tgtaggtatc tcagttcggt g
//
