LOCUS       sequence-237144-        5138 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 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.Intron9.CMV/CB-EGFP
FEATURES             Location/Qualifiers
     source          1..5138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        83..331
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     83..103
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     254..273
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        353..428
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        442..821
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1117..1135
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1158..1160,1161..1163,1164..1877)
                     /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(1158..1160,1161..1163,1164..1874)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1203..1224)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1464..1483)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1811..1832
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1960..1979)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2025..2080
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   2128..2257
                     /label=AAV2 ITR
     primer_bind     complement(2276..2293)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2276..2292)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2285..2307)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2434..2889
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2521..2540)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2731..2752
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2915..3019
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3020..3088,3089..3880)
                     /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(3238..3257)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4051..4639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4540..4559
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4927..4948
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4963..4968,4969..4986,4987..4993)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5001..5017
                     /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     5006..5028
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5025..5041
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5025..5041
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgtagcc atgctctagg
       61 aagatcaatt caattcacgc gtgagggcct atttcccatg attccttcat atttgcatat
      121 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      181 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      241 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      301 tttatatatc ttgtggaaag gacgaaacac cgcattgcac aaaacaaaat gcgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
      421 gtcggtgctt ttttacgcgt cgacattgat tattgactag ttattaatag taatcaatta
      481 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      541 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      601 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      661 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      721 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      781 cttggcagta catctacgta ttagtcatcg ctattaccat gtcgaggcca cgttctgctt
      841 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
      901 attttgtgca gcgatggggg cggggggggg gggcgcgcgc caggcggggc ggggcggggc
      961 gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc
     1021 cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg
     1081 cggcgggcgg gagcaagctc tagcctcgag aatacttaat acgactcact ataggctagt
     1141 aatacgactc actatagatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1201 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1261 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1321 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1381 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1441 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1501 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1561 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1621 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1681 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1741 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1801 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1861 acgagctgta caagtaaagc ggccctagcg tttaaacggg ccctctagag tcgacccggg
     1921 cggcctcgag gacggggtga actacgcctg aggatccgat ctttttccct ctgccaaaaa
     1981 ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat
     2041 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggcctag gtagataagt
     2101 agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct
     2161 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     2221 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagcct taattaacct aattcactgg
     2281 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     2341 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     2401 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2461 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2521 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2581 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2641 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2701 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2761 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2821 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     2881 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2941 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3001 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3061 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3121 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3181 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3241 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3301 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3361 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3421 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3481 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3541 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3601 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3661 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3721 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3781 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3841 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3901 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3961 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4021 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4081 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4141 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4201 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4261 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4321 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4381 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4441 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4501 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4561 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4621 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4681 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4741 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4801 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     4861 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     4921 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     4981 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     5041 ccatgattac gccagattta attaaggcct taattaggct gcgcgctcgc tcgctcactg
     5101 aggccgcccg ggcaaagccc gggcgtcggg cgaccttt
//
