LOCUS       sequence-304586-        7378 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hu6 HGPS sgRNA expression and ABE7.10max VRQR C terminal AAV vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7378)
  AUTHORS   Koblan LW, Erdos MR, Wilson C, Cabral WA, Levy JM, Xiong ZM, Tavarez
            UL, Davison LM, Gete YG, Mao X, Newby GA, Doherty SP, Narisu N, 
            Sheng Q, Krilow C, Lin CY, Gordon LB, Cao K, Collins FS, Brown JD, 
            Liu DR
  TITLE     In vivo base editing rescues Hutchinson-Gilford progeria syndrome in
            mice.
  JOURNAL   Nature. 2021 Jan;589(7843):608-614. doi: 10.1038/s41586-020-03086-7.
            Epub 2021 Jan 6.
  PUBMED    33408413
REFERENCE   2  (bases 1 to 7378)
  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/s41586-020-03086-7"; journalName: "Nature"; date: 
            "2021-01"; volume: "589"; issue: "7843"; pages: "608-614"
COMMENT     Sequence Label: hu6 HGPS sgRNA expression and ABE7.10max VRQR ...
FEATURES             Location/Qualifiers
     source          1..7378
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..286
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        288..565
                     /label=chicken beta-actin promoter
     intron          566..793
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             847..867
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             973..3357
                     /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
                     QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
                     FDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             3400..3420
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3485..3505)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3681..3905
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(3919..3994)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4015..4263)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4073..4092)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4243..4263)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4287..4416
                     /label=AAV2 ITR
     primer_bind     complement(4435..4452)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4435..4451)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4444..4466)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4593..5048
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4680..4699)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4890..4911
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5074..5178
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5179..5247,5248..6039)
                     /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(5397..5416)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6210..6798
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6699..6718
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7086..7107
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7122..7127,7128..7145,7146..7152)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7160..7176
                     /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     7165..7187
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7184..7200
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7184..7200
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   7227..7356
                     /label=AAV2 ITR
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      121 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      181 cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt
      241 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      301 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      361 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg
      421 cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa
      481 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      541 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
      601 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
      661 gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt
      721 taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat
      781 cacttttttt caggttggac cggtgccacc atgaaacgga cagccgacgg aagcgagttc
      841 gagtcaccaa agaagaagcg gaaagtcatc aagattgcta cacggaaata cctgggaaag
      901 cagaacgtgt acgacatcgg cgtggagcgg gatcacaact tcgccctgaa gaatggcttt
      961 atcgccagca attgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     1021 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     1081 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     1141 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     1201 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     1261 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     1321 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     1381 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     1441 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     1501 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     1561 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     1621 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     1681 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     1741 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac
     1801 gactccatcg ataacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
     1861 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
     1921 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     1981 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     2041 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
     2101 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     2161 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
     2221 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
     2281 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
     2341 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     2401 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc
     2461 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
     2521 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     2581 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
     2641 aagtacggcg gcttcgtgag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     2701 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     2761 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     2821 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     2881 cggaagagaa tgctggcctc agccagagaa ctgcagaagg gaaacgaact ggccctgccc
     2941 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
     3001 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc
     3061 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa
     3121 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
     3181 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
     3241 accatcgacc ggaagcagta cagaagcacc aaagaggtgc tggacgccac cctgatccac
     3301 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggtgactct
     3361 ggcggctcaa aaagaaccgc cgacggcagc gaattcgagc ccaagaagaa gaggaaagtc
     3421 taagatctga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3481 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3541 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggtta gttcttgcca
     3601 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     3661 ctgacaattc cgtggtgcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3721 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3781 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3841 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3901 tatggctcga gaaaaaaagc accgactcgg tgccactttt tcaagttgat aacggactag
     3961 ccttatttta acttgctatt tctagctcta aaacggagcc caggtgggtg gaccggtgtt
     4021 tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag
     4081 catatgatag tccattttaa aacataattt taaaactgca aactacccaa gaaattatta
     4141 ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattctaat
     4201 tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc
     4261 ctcttcctgc ccgaccttgc ggccgcagga acccctagtg atggagttgg ccactccctc
     4321 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     4381 tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta attcactggc
     4441 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     4501 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     4561 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     4621 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4681 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4741 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4801 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4861 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4921 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4981 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     5041 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5101 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5161 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5221 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5281 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5341 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5401 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5461 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5521 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5581 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5641 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5701 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5761 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5821 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5881 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5941 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6001 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6061 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6121 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6181 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6241 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6301 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6361 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6421 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6481 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6541 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6601 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6661 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6721 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6781 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6841 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     6901 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     6961 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     7021 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     7081 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     7141 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     7201 catgattacg ccagatttaa ttaaggctgc gcgctcgctc gctcactgag gccgcccggg
     7261 caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca
     7321 gagagggagt ggccaactcc atcactaggg gttcctgcgg cctctagatc agggtacc
//
