LOCUS       sequence-234592-        4886 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sgRNA targeting the intron in the MADM alleles (GT and 
            TG)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4886)
  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 4886)
  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-sgGTTG.CMV/CB-Gluc
FEATURES             Location/Qualifiers
     source          1..4886
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     136..155
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        234..309
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        323..702
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     regulatory      992..1001
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             998..1555
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     primer_bind     complement(1591..1610)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1656..1711
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   1759..1888
                     /label=AAV2 ITR
     primer_bind     complement(1907..1924)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1907..1923)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1916..1938)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2065..2520
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2152..2171)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2362..2383
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2546..2650
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2651..2719,2720..3511)
                     /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(2869..2888)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3682..4270
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4171..4190
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4558..4579
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4594..4599,4600..4617,4618..4624)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4632..4648
                     /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     4637..4659
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4656..4672
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4656..4672
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        join(4851..4886,1..213)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4851..4871
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
       61 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      121 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      181 gctttatata tcttgtggaa aggacgaaac accgggtggg aaaatagacc aatgttttag
      241 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      301 agtcggtgct tttttacgcg tcgacattga ttattgacta gttattaata gtaatcaatt
      361 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      421 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      481 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      541 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      601 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      661 acttggcagt acatctacgt attagtcatc gctattacca tgtcgaggcc acgttctgct
      721 tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt attttttaat
      781 tattttgtgc agcgatgggg gcgggggggg ggggcgcgcg ccaggcgggg cggggcgggg
      841 cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct
      901 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc
      961 gcggcgggcg ggagcaagct ctagcctcga ggccaccatg ggagtcaaag ttctgtttgc
     1021 cctgatctgc atcgctgtgg ccgaggccaa gcccaccgag aacaacgagg acttcaacat
     1081 cgtggccgtg gccagcaact tcgcgaccac ggatctcgat gctgaccgcg ggaagttgcc
     1141 cggcaagaag ctgccgctgg aggtgctcaa agagatggaa gccaatgccc ggaaagctgg
     1201 ctgcaccagg ggctgtctga tctgcctgtc ccacatcaag tgcacgccca agatgaagaa
     1261 gttcatccca ggacgctgcc acacctacga aggcgacaaa gagtccgcac agggcggcat
     1321 aggcgaggcg atcgtcgaca ttcctgagat tcctgggttc aaggacttgg agcccatgga
     1381 gcagttcatc gcacaggtcg atctgtgtgt ggactgcaca actggctgcc tcaaagggct
     1441 tgccaacgtg cagtgttctg acctgctcaa gaagtggctg ccgcaacgct gtgcgacctt
     1501 tgccagcaag atccagggcc aggtggacaa gatcaagggg gccggtggtg actagctcga
     1561 ggacggggtg aactacgcct gaggatccga tctttttccc tctgccaaaa attatgggga
     1621 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     1681 aatagtgtgt tggaattttt tgtgtctctc actcggccta ggtagataag tagcatggcg
     1741 ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     1801 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     1861 cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc taattcactg gccgtcgttt
     1921 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     1981 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2041 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2101 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2161 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2221 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2281 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2341 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2401 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2461 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2521 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2581 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2641 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2701 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2761 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2821 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2881 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2941 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3001 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3061 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3121 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3181 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3241 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3301 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3361 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3421 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3481 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3541 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3601 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3661 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3721 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3781 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3841 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3901 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3961 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4021 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4081 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4141 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4201 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4261 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4321 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4381 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4441 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     4501 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     4561 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     4621 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     4681 cgccagattt aattaaggcc ttaattaggc tgcgcgctcg ctcgctcact gaggccgccc
     4741 gggcaaagcc cgggcgsscy crggcaaagc ccgggcggcc tcagtgagcg agcgagcgcg
     4801 cagagaggga gtgtagccat gctctaggaa gatcaattca attcacgcgt gagggcctat
     4861 ttcccatgat tccttcatat ttgcat
//
