LOCUS       sequence-330342-        5982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid used for Crispr/cas9 based disruption of mouse Trim28, guide
            targeting exon 13. Expressing nuclear RFP. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5982)
  AUTHORS   Jonsson ME, Garza R, Sharma Y, Petri R, Sodersten E, Johansson JG, 
            Johansson PA, Atacho DA, Pircs K, Madsen S, Yudovich D, Ramakrishnan
            R, Holmberg J, Larsson J, Jern P, Jakobsson J
  TITLE     Activation of endogenous retroviruses during brain development 
            causes an inflammatory response.
  JOURNAL   EMBO J. 2021 Mar 1:e106423. doi: 10.15252/embj.2020106423.
  PUBMED    33644903
REFERENCE   2  (bases 1 to 5982)
  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"; journalName: "EMBO J. 
            2021 Mar 1:e106423. doi: 10.15252/embj.2020106423."
COMMENT     Sequence Label: pAAV.U6gT28.13.hSyn.H2B.RFP
FEATURES             Location/Qualifiers
     source          1..5982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     358..377
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        457..532
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        572..1019
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1056..1433
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      1446..1455
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1452..2162
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1597..1615)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1865..1884
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2181..2769
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2234..2254)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2652..2663)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2817..2951
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2854..2873)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2908..2927
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3388..3976)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3468..3487)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4147..4938,4939..5007))
                     /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     4770..4789
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5008..5112)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5180..5198
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5236..5258)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5358..5377
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5444..5899)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5581..5602)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5793..5812
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gggggggggg ggggggggtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
       61 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
      121 gcgcgcagag agggagtggc caactccatc actaggggtt cctcagatcg ggctgcagga
      181 attggccgct ctagatttcc catgattcct tcatatttgc atatacgata caaggctgtt
      241 agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      301 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      361 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      421 aaaggacgaa acaccgcgca ctggttacac atgaccgttt tagagctaga aatagcaagt
      481 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc
      541 tagactctag ctgcagaggg acctgcgtat gagtgcaagt gggttttagg accaggatga
      601 ggcggggtgg gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc
      661 ccattcccca aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag
      721 gcgcgtgcgc actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg
      781 cgcgccaccg ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa
      841 ctccccttcc cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc
      901 acgcgaggcg cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact
      961 cagcgctgcc tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcgcagt
     1021 cgaattcaag ctgctagcag cttggtaccg ccaccatgcc tgaaccctct aagtctgctc
     1081 cagcccctaa aaagggttct aagaaggcta tcactaaggc gcagaagaag gatggtaaga
     1141 agcgtaagcg cagccgcaag gagagctatt ctatctatgt gtacaaggtt ctgaagcagg
     1201 tccaccccga caccggcatc tcatccaagg ccatggggat catgaactcc ttcgtcaacg
     1261 acatcttcga gcgcatcgcg ggcgaggctt ctcgcctggc tcactacaat aagcgctcga
     1321 ccatcacctc cagggagatt cagacggctg tgcgcctgct gctgcctggg gagctggcta
     1381 agcatgctgt gtccgagggc actaaggcag ttaccaagta cactagctct aaggatccac
     1441 cggtcgccac catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
     1501 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     1561 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     1621 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     1681 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     1741 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     1801 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     1861 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     1921 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     1981 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     2041 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     2101 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     2161 aaactagtgg atccgtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg
     2221 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     2281 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     2341 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     2401 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     2461 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     2521 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga
     2581 cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     2641 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     2701 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     2761 cctccccgcc tggaattcga gctcggtaca gcttatcgat accgtcgact tcgagcaact
     2821 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     2881 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     2941 atgtctggat cgtctagcat cgaagatccc ccgatctgag gaacccctag tgatggagtt
     3001 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg
     3061 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc
     3121 caaccccccc cccccccccc ctgcagccca gctgcattaa tgaatcggcc aacgcgcggg
     3181 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3241 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3301 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3361 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3421 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3481 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3541 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta
     3601 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3661 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3721 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3781 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     3841 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3901 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3961 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4021 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4081 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4141 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4201 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4261 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4321 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4381 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4441 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4501 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4561 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4621 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4681 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4741 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4801 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4861 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4921 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4981 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5041 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5101 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     5161 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     5221 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     5281 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     5341 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
     5401 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aaacgttaat
     5461 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     5521 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5581 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     5641 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     5701 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     5761 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     5821 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     5881 gcgccgctac agggcgcgtc gcgccattcg ccattcaggc tacgcaactg ttgggaaggg
     5941 cgatcggtgc gggcctcttc gctattacgc cagctggctg ca
//
