LOCUS       sequence-234590-        5138 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting mouse Idua intron 9 (sgIdua)..
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-sgIdua.CMV/CB-EGFP
FEATURES             Location/Qualifiers
     source          1..5138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        61..309
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     61..81
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     232..251
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        331..406
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        420..799
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1095..1113
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1136..1138,1139..1141,1142..1855)
                     /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(1136..1138,1139..1141,1142..1852)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1181..1202)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1442..1461)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1789..1810
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1938..1957)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2003..2058
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   2106..2235
                     /label=AAV2 ITR
     primer_bind     complement(2254..2271)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2254..2270)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2263..2285)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2412..2867
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2499..2518)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2709..2730
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2893..2997
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2998..3066,3067..3858)
                     /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(3216..3235)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4029..4617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4518..4537
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4905..4926
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4941..4946,4947..4964,4965..4971)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4979..4995
                     /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     4984..5006
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5003..5019
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5003..5019
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 agcgagcgcg cagagaggga gtgtagccat gctctaggaa gatcaattca attcacgcgt
       61 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
      121 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      181 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      241 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      301 cgaaacaccg tgggagctag atattagggt gttttagagc tagaaatagc aagttaaaat
      361 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttacgcgtcg
      421 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      481 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      541 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      601 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      661 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      721 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      781 agtcatcgct attaccatgt cgaggccacg ttctgcttca ctctccccat ctcccccccc
      841 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg
      901 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
      961 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
     1021 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gcaagctcta
     1081 gcctcgagaa tacttaatac gactcactat aggctagtaa tacgactcac tatagatggt
     1141 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1201 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1261 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1321 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1381 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1441 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1501 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1561 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1621 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1681 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1741 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1801 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg
     1861 ccctagcgtt taaacgggcc ctctagagtc gacccgggcg gcctcgagga cggggtgaac
     1921 tacgcctgag gatccgatct ttttccctct gccaaaaatt atggggacat catgaagccc
     1981 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg
     2041 aattttttgt gtctctcact cggcctaggt agataagtag catggcgggt taatcattaa
     2101 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     2161 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     2221 cgagcgagcg cgcagcctta attaacctaa ttcactggcc gtcgttttac aacgtcgtga
     2281 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     2341 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     2401 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2461 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2521 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2581 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2641 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2701 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2761 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2821 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     2881 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2941 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3001 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3061 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3121 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3181 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3241 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3301 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     3361 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     3421 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     3481 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3541 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3601 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3661 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3721 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3781 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3841 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3901 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3961 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4021 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4081 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4141 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4201 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4261 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4321 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4381 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4441 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4501 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4561 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4621 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4681 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4741 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4801 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     4861 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     4921 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     4981 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc cagatttaat
     5041 taaggcctta attaggctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg
     5101 gcgtcgggcg acctttggtc gcccggcctc agtgagcg
//
