LOCUS       sequence-231443-        5144 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting mouse Fah intron 9 (sgFah.Intron9)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5144)
  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 5144)
  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.Intron9.CMV/CB-EGFP
FEATURES             Location/Qualifiers
     source          1..5144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        148..396
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     148..168
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     319..338
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        418..493
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        507..886
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1183..1201
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1224..1226,1227..1229,1230..1943)
                     /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(1224..1226,1227..1229,1230..1940)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1269..1290)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1530..1549)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1877..1898
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2026..2045)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2091..2146
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   2194..2323
                     /label=AAV2 ITR
     primer_bind     complement(2342..2359)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2342..2358)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2351..2373)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2500..2955
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2587..2606)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2797..2818
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2981..3085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3086..3154,3155..3946)
                     /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(3304..3323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4117..4705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4606..4625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4993..5014
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5029..5034,5035..5052,5053..5059)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5067..5083
                     /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     5072..5094
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5091..5107
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5091..5107
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgtagcc atgctctagg
      121 aagatcaatt cggtacaatt cacgcgtgag ggcctatttc ccatgattcc ttcatatttg
      181 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
      241 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
      301 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
      361 ttggctttat atatcttgtg gaaaggacga aacaccgcat tgcacaaaac aaaatgcgtt
      421 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      481 accgagtcgg tgctttttta cgcgtcgaca ttgattattg actagttatt aatagtaatc
      541 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      601 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      661 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      721 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      781 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      841 tcctacttgg cagtacatct acgtattagt catcgctatt accatgtcga ggccacgttc
      901 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
      961 taattatttt gtgcagcgat gggggcgggg ggggggggcg cgcgccaggc ggggcggggc
     1021 ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg
     1081 cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa
     1141 gcgcgcggcg ggcgggagca agctctagcc tcgagaatta cttaatacga ctcactatag
     1201 gctagtaata cgactcacta tagatggtga gcaagggcga ggagctgttc accggggtgg
     1261 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1321 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1381 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1441 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1501 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1561 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1621 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1681 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     1741 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1801 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     1861 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1921 gcatggacga gctgtacaag taaagcggcc ctagcgttta aacgggccct ctagagtcga
     1981 cccgggcggc ctcgaggacg gggtgaacta cgcctgagga tccgatcttt ttccctctgc
     2041 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     2101 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gcctaggtag
     2161 ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca
     2221 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     2281 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat taacctaatt
     2341 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     2401 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     2461 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     2521 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2581 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2641 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2701 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2761 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2821 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2881 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     2941 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3001 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3061 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3121 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3181 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3241 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3301 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     3361 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3421 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3481 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3541 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3601 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     3661 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3721 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3781 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3841 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3901 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3961 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4021 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4081 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4141 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4201 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4261 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4321 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4381 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4441 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4501 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4561 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4621 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4681 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4741 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4801 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4861 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     4921 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     4981 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     5041 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     5101 ctatgaccat gattacgcca gatttaatta aggccttaat tagg
//
