LOCUS       sequence-234582-        5137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting mouse Aspa intron 2 (sgAspa)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5137)
  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 5137)
  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-sgAspa.CMV/CB-EGFP
FEATURES             Location/Qualifiers
     source          1..5137
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        116..364
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     116..136
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     287..306
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        385..460
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        474..853
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1149..1167
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1190..1192,1193..1195,1196..1909)
                     /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(1190..1192,1193..1195,1196..1906)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1235..1256)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1496..1515)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1843..1864
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1992..2011)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2057..2112
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   2160..2289
                     /label=AAV2 ITR
     primer_bind     complement(2308..2325)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2308..2324)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2317..2339)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2466..2921
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2553..2572)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2763..2784
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2947..3051
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3052..3120,3121..3912)
                     /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(3270..3289)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4083..4671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4572..4591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4959..4980
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4995..5000,5001..5018,5019..5025)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5033..5049
                     /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     5038..5060
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5057..5073
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5057..5073
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
       61 gcgcgcagag agggagtgta gccatgctct aggaagatca attcaattca cgcgtgaggg
      121 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
      181 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
      241 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
      301 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
      361 caccggtatt gtacaaagat acaggtttta gagctagaaa tagcaagtta aaataaggct
      421 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttacgc gtcgacattg
      481 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      541 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      601 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      661 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      721 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      781 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      841 cgctattacc atgtcgaggc cacgttctgc ttcactctcc ccatctcccc cccctcccca
      901 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
      961 gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag
     1021 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
     1081 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagcaagc tctagcctcg
     1141 agaatactta atacgactca ctataggcta gtaatacgac tcactataga tggtgagcaa
     1201 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1261 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1321 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1381 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     1441 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     1501 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1561 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     1621 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     1681 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     1741 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     1801 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     1861 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccctag
     1921 cgtttaaacg ggccctctag agtcgacccg ggcggcctcg aggacggggt gaactacgcc
     1981 tgaggatccg atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     2041 catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt
     2101 ttgtgtctct cactcggcct aggtagataa gtagcatggc gggttaatca ttaactacaa
     2161 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     2221 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     2281 agcgcgcagc cttaattaac ctaattcact ggccgtcgtt ttacaacgtc gtgactggga
     2341 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     2401 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2461 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2521 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2581 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2641 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2701 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2761 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2821 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2881 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     2941 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3001 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3061 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3121 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3181 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3241 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     3301 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3361 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3421 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3481 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3541 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     3601 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3661 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3721 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3781 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3841 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3901 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3961 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4021 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4081 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4141 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4201 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4261 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4321 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4381 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4441 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4501 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4561 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4621 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4681 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4741 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4801 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     4861 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     4921 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     4981 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     5041 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccagatt taattaaggc
     5101 cttaattagg ctgcgcgctc gctcgctcac tgaggcc
//
