LOCUS       sequence-234575-        5138 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting mouse Fah intron 7 (sgFah)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5138)
  AUTHORS   Wang D, Li J, Song CQ, Tran K, Mou H, Wu PH, Tai PWL, Mendonca CA, 
            Ren L, Wang BY, Su Q, Gessler DJ, Zamore PD, Xue W, Gao G
  TITLE     Cas9-mediated allelic exchange repairs compound heterozygous 
            recessive mutations in mice.
  JOURNAL   Nat Biotechnol. 2018 Oct;36(9):839-842. doi: 10.1038/nbt.4219. Epub 
            2018 Aug 13.
  PUBMED    30102296
REFERENCE   2  (bases 1 to 5138)
  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/nbt.4219"; 
            journalName: "Nat Biotechnol"; date: "2018-10"; volume: "36"; issue:
            "9"; pages: "839-842"
COMMENT     Sequence Label: pAAVsc.U6-sgFah.CMV/CB-EGFP
FEATURES             Location/Qualifiers
     source          1..5138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     160..179
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        259..334
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        348..727
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1023..1041
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1064..1066,1067..1069,1070..1783)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1064..1066,1067..1069,1070..1780)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1109..1130)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1370..1389)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1717..1738
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1866..1885)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1931..1986
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   2034..2163
                     /label=AAV2 ITR
     primer_bind     complement(2182..2199)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2182..2198)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2191..2213)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2340..2795
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2427..2446)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2637..2658
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2821..2925
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2926..2994,2995..3786)
                     /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(3144..3163)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3957..4545
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4446..4465
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4833..4854
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4869..4874,4875..4892,4893..4899)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4907..4923
                     /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     4912..4934
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4931..4947
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4931..4947
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        join(5127..5138,1..237)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     join(5127..5138,1..9)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
       61 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
      121 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
      181 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgaa
      241 agagtggagc cttagtgtgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
      301 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt acgcgtcgac attgattatt
      361 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      421 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      481 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      721 taccatgtcg aggccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca
      781 attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggc
      841 gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg
      901 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
      961 ggcggcccta taaaaagcga agcgcgcggc gggcgggagc aagctctagc ctcgagaata
     1021 cttaatacga ctcactatag gctagtaata cgactcacta tagatggtga gcaagggcga
     1081 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     1141 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     1201 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     1261 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     1321 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     1381 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     1441 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     1501 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     1561 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     1621 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     1681 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     1741 cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc ctagcgttta
     1801 aacgggccct ctagagtcga cccgggcggc ctcgaggacg gggtgaacta cgcctgagga
     1861 tccgatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     1921 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     1981 ctctcactcg gcctaggtag ataagtagca tggcgggtta atcattaact acaaggaacc
     2041 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     2101 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     2161 cagccttaat taacctaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     2221 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     2281 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     2341 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2401 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2461 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2521 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2581 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2641 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2701 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2761 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     2821 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2881 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     2941 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3001 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3061 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3121 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     3181 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3241 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3301 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3361 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3421 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3481 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3541 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3601 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3661 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3721 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3781 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3841 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3901 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3961 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4021 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4081 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4141 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4201 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4261 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4321 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4381 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4441 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4501 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4561 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4621 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4681 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     4741 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     4801 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     4861 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     4921 caatttcaca caggaaacag ctatgaccat gattacgcca gatttaatta aggccttaat
     4981 taggctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac
     5041 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgt agccatgctc
     5101 taggaagatc aattcaattc acgcgtgagg gcctattt
//
