LOCUS       sequence-456168-        7652 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses the C-terminal of AAV-NG-CBE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7652)
  AUTHORS   Liu Z, Chen S, Davis AE, Lo CH, Wang Q, Li T, Ning K, Zhang Q, Zhao 
            J, Wang S, Sun Y
  TITLE     Efficient Rescue of Retinal Degeneration in Pde6a Mice by Engineered
            Base Editing and Prime Editing.
  JOURNAL   Adv Sci (Weinh). 2024 Sep 19:e2405628. doi: 10.1002/advs.202405628.
  PUBMED    39297417
REFERENCE   2  (bases 1 to 7652)
  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: "Adv Sci 
            (Weinh). 2024 Sep 19:e2405628. doi: 10.1002/advs.202405628."
COMMENT     Sequence Label: AAV-NG-CBE C
FEATURES             Location/Qualifiers
     source          1..7652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..38
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(53..58,59..76,77..83)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    91..107
                     /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     96..118
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     115..131
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     115..131
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   158..287
                     /label=AAV2 ITR
     enhancer        310..595
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          875..1102
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1156..1176
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1282..3666
                     /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
                     QVNIVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKY
                     FDTTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             3697..3945
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3982..4002
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4067..4087)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4263..4487
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(4501..4576)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4598..4846)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4656..4675)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4826..4846)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4870..4999
                     /label=AAV2 ITR
     primer_bind     complement(5018..5035)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5018..5034)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5027..5049)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5176..5631
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5263..5282)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5473..5494
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5657..5761
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5762..5830,5831..6622)
                     /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(5980..5999)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6793..7381
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7282..7301
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
       61 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
      121 acagctatga ccatgattac gccagattta attaaggctg cgcgctcgct cgctcactga
      181 ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga
      241 gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg gcctctagat
      301 cagggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      361 ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc aatgggtgga
      421 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      481 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt acatgacctt
      541 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga
      601 ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt
      661 gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg
      721 cgcgccgggg gggggggggg ggggsggggg gsggggsggg gsgrggsggr gaggkgcggc
      781 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
      841 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
      901 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
      961 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc tgagcaagag
     1021 gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc tggagcacct
     1081 gcctgaaatc actttttttc aggttggacc ggtgccacca tgaaacggac agccgacgga
     1141 agcgagttcg agtcaccaaa gaagaagcgg aaagtcatca agattgctac acggaaatac
     1201 ctgggaaagc agaacgtgta cgacatcggc gtggagcggg atcacaactt cgccctgaag
     1261 aatggcttta tcgccagcaa ttgcttcgac tccgtggaaa tctccggcgt ggaagatcgg
     1321 ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc
     1381 ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt
     1441 gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa
     1501 gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg
     1561 atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac
     1621 ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag
     1681 gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat
     1741 ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag
     1801 ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag
     1861 aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag
     1921 ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg
     1981 cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag
     2041 gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt
     2101 ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag
     2161 agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg
     2221 ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc
     2281 ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag
     2341 atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat
     2401 gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc
     2461 cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac
     2521 gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc
     2581 gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag
     2641 caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc
     2701 aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac
     2761 ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg
     2821 ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc
     2881 aaagagtcta tccggcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg
     2941 gaccctaaga agtacggcgg cttcgtgagc cccaccgtgg cctattctgt gctggtggtg
     3001 gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc
     3061 accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc
     3121 tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg
     3181 gaaaacggcc ggaagagaat gctggcctct gcccggttcc tgcagaaggg aaacgaactg
     3241 gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag
     3301 ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg
     3361 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat
     3421 ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc
     3481 gagaatatca tccacctgtt taccctgacc aatctgggag cccctcgggc cttcaagtac
     3541 tttgacacca ccatcgaccg gaaggtgtac cggagcacca aagaggtgct ggacgccacc
     3601 ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga
     3661 ggtgacagcg gcgggagcgg cgggagcggg gggagcacta atctgagcga catcattgag
     3721 aaggagactg ggaaacagct ggtcattcag gagtccatcc tgatgctgcc tgaggaggtg
     3781 gaggaagtga tcggcaacaa gccagagtct gacatcctgg tgcacaccgc ctacgacgag
     3841 tccacagatg agaatgtgat gctgctgacc tctgacgccc ccgagtataa gccttgggcc
     3901 ctggtcatcc aggattctaa cggcgagaat aagatcaaga tgctgggatc caaaagaacc
     3961 gccgacggca gcgaattcga gcccaagaag aagaggaaag tctaagatct gataatcaac
     4021 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     4081 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     4141 tcattttctc ctccttgtat aaatcctggt tagttcttgc cacggcggaa ctcatcgccg
     4201 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgc
     4261 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     4321 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     4381 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4441 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctc gagaaaaaaa
     4501 gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta
     4561 tttctagctc taaaaccgag acgcattaat cgtctcgggt gtttcgtcct ttccacaaga
     4621 tatataaagc caagaaatcg aaatactttc aagttacggt aagcatatga tagtccattt
     4681 taaaacataa ttttaaaact gcaaactacc caagaaatta ttactttcta cgtcacgtat
     4741 tttgtactaa tatctttgtg tttacagtca aattaattct aattatctct ctaacagcct
     4801 tgtatcgtat atgcaaatat gaaggaatca tgggaaatag gccctcttcc tgcccgacct
     4861 tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     4921 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     4981 tgagcgagcg agcgcgcagc cttaattaac ctaattcact ggccgtcgtt ttacaacgtc
     5041 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     5101 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     5161 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     5221 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5281 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5341 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5401 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5461 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5521 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5581 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5641 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5701 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5761 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5821 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5881 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5941 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6001 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6061 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6121 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6181 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6241 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6301 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6361 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6421 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6481 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6541 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6601 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6661 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6721 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6781 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6841 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6901 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6961 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7021 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7081 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7141 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7201 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7261 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7321 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7381 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7441 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7501 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     7561 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     7621 gcacgacagg tttcccgact ggaaagcggg ca
//
