LOCUS       sequence-489035-        7131 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CBh promoter expression plasmid for N-terminal intein-split AAV 
            construct with N-term of ABE8e-SpCas9(S55R) - to be used with eVRQR 
            constructs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7131)
  AUTHORS   Alves CRR, Das S, Krishnan V, Ha LL, Fox LR, Stutzman HE, Shamber 
            CE, Kalailingam P, McCarthy S, Lino Cardenas CL, Fong CE, Imai T, 
            Mitra S, Yun S, Wood RK, Benning FMC, Roh K, Lawton J, Kim N, 
            Silverstein RA, Ferreira da Silva J, de la Cruz D, Richa R, Xie J, 
            Gray-Edwards HL, Malhotra R, Chung DY, Chao LH, Tsai SQ, Maguire CA,
            Lindsay ME, Kleinstiver BP, Musolino PL
  TITLE     Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 
            base editor in mice.
  JOURNAL   Nat Biomed Eng. 2025 Sep 11:10.1038/s41551-025-01499-1. doi: 
            10.1038/s41551-025-01499-1.
  PUBMED    40935887
REFERENCE   2  (bases 1 to 7131)
  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 Biomed 
            Eng. 2025 Sep 11:10.1038/s41551-025-01499-1. doi: 
            10.1038/s41551-025-01499-1."
COMMENT     Sequence Label: N-term AAV-ABE8e-SpCas9(S55R) (CA21)
FEATURES             Location/Qualifiers
     source          1..7131
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(125..142)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(125..141)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(134..156)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      283..738
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(370..389)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     580..601
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        764..868
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(869..937,938..1729)
                     /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     complement(1087..1106)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1900..2488
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2389..2408
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2776..2797
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2812..2817,2818..2835,2836..2842)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2850..2866
                     /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)."
     primer_bind     2855..2877
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2874..2890
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2874..2890
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2917..3046
                     /label=AAV2 ITR
     enhancer        3069..3354
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        3356..3633
                     /label=chicken beta-actin promoter
     intron          3634..3861
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3915..3935
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4530..6245
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDRGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             6594..6614
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(6679..6699)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    6875..7099
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   join(7108..7131,1..106)
                     /label=AAV2 ITR
ORIGIN
        1 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
       61 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta
      121 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
      181 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
      241 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
      301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
      541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
      601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
      661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
      721 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      781 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      841 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      901 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      961 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1021 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1081 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1141 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1201 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1261 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1321 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1381 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1441 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1501 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1561 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1621 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1681 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1741 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1801 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1861 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1921 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1981 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2041 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2101 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2161 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2221 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2281 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2341 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2401 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2461 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2521 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2581 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2641 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     2701 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2761 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     2821 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     2881 cagctatgac catgattacg ccagatttaa ttaaggctgc gcgctcgctc gctcactgag
     2941 gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     3001 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagatc
     3061 agggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     3121 cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca atgggtggag
     3181 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     3241 cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta catgacctta
     3301 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag
     3361 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg
     3421 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc
     3481 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
     3541 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     3601 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
     3661 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
     3721 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg
     3781 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg
     3841 cctgaaatca ctttttttca ggttggaccg gtgccaccat gaaacggaca gccgacggaa
     3901 gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt tcccacgagt
     3961 actggatgag acatgccctg accctggcca agagggcacg cgatgagagg gaggtgcctg
     4021 tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac agagccatcg
     4081 gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc ggcctggtca
     4141 tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct tgcgtgatgt
     4201 gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg aggaacagca
     4261 aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg aatcaccgcg
     4321 tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc gatttctacc
     4381 ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc aactccggag
     4441 gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc gcaacacctg
     4501 aaagcagcgg gggcagcagc ggggggtcag acaagaagta cagcatcggc ctggccatcg
     4561 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     4621 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     4681 tgttcgacag aggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     4741 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     4801 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     4861 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     4921 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     4981 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     5041 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     5101 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     5161 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     5221 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     5281 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     5341 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     5401 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     5461 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     5521 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     5581 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     5641 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     5701 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     5761 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     5821 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     5881 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     5941 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     6001 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     6061 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     6121 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     6181 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     6241 tcgagtgcct gtcctacgag acagagatcc tgacagtgga gtatggcctg ctgccaatcg
     6301 gcaagatcgt ggagaagagg atcgagtgta ccgtgtactc tgtggataac aatggcaaca
     6361 tctatacaca gcccgtggca cagtggcacg ataggggaga gcaggaggtg ttcgagtatt
     6421 gcctggagga cggcagcctg atcagggcaa ccaaggacca caagttcatg acagtggatg
     6481 gccagatgct gcccatcgac gagattttcg agcgggagct ggacctgatg agagtggata
     6541 acctgcctaa tagcggaggc agtaaaagaa cagcagacgg gagtgagttt gagcccaaga
     6601 aaaagagaaa ggtgtaagat ctgataatca acctctggat tacaaaattt gtgaaagatt
     6661 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     6721 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     6781 gttagttctt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     6841 tcggctgttg ggcactgaca attccgtggt gcgactgtgc cttctagttg ccagccatct
     6901 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     6961 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     7021 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     7081 gatgcggtgg gctctatggg cggccgcagg aacccctagt gatggagttg g
//
