LOCUS       sequence-330339-        6342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid used for Crispr/cas9 based disruption of mouse Trim28, guide
            targeting exon 4. Cre-inducible expression of nuclear RFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6342)
  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 6342)
  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.U6gT28.4.hSyn.flex.H2B.RFP
FEATURES             Location/Qualifiers
     source          1..6342
                     /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"
     protein_bind    complement(1129..1162)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1213..1246)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1263..1973)
                     /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(1541..1560)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1810..1828
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1970..1979
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1992..2369)
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     protein_bind    2399..2432
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2483..2516
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2541..3129
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2594..2614)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3012..3023)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3177..3311
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3214..3233)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3268..3287
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3748..4336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3828..3847)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4507..5298,5299..5367))
                     /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     5130..5149
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5368..5472)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5540..5558
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5596..5618)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5718..5737
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5804..6259)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5941..5962)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6153..6172
                     /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 acaccgggtc cttgtgagcg ttgagcgttt 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 gccggcccag
      961 ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat ctgcgctgcg
     1021 gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc gtgtcgtgcc
     1081 tgagagcgca gtcgagaagg taccggatcc tctagagtcg actccggaat aacttcgtat
     1141 aggatacttt atacgaagtt atgcagaatg gtagctggat tgtagctgct attagcaata
     1201 tgaaacctct taataacttc gtatagcata cattatacga agttatggcg cgccatacta
     1261 gtttacttgt acagctcgtc catgccgccg gtggagtggc ggccctcggc gcgttcgtac
     1321 tgttccacga tggtgtagtc ctcgttgtgg gaggtgatgt ccaacttgat gttgacgttg
     1381 taggcgccgg gcagctgcac gggcttcttg gccttgtagg tggtcttgac ctcagcgtcg
     1441 tagtggccgc cgtccttcag cttcagcctc tgcttgatct cgcccttcag ggcgccgtcc
     1501 tcggggtaca tccgctcgga ggaggcctcc cagcccatgg tcttcttctg cattacgggg
     1561 ccgtcggagg ggaagttggt gccgcgcagc ttcaccttgt agatgaactc gccgtcctgc
     1621 agggaggagt cctgggtcac ggtcaccacg ccgccgtcct cgaagttcat cacgcgctcc
     1681 cacttgaagc cctcggggaa ggacagcttc aagtagtcgg ggatgtcggc ggggtgcttc
     1741 acgtaggcct tggagccgta catgaactga ggggacagga tgtcccaggc gaagggcagg
     1801 gggccaccct tggtcacctt cagcttggcg gtctgggtgc cctcgtaggg gcggccctcg
     1861 ccctcgccct cgatctcgaa ctcgtggccg ttcacggagc cctccatgtg caccttgaag
     1921 cgcatgaact ccttgatgat ggccatgtta tcctcctcgc ccttgctcac catggtggcg
     1981 accggtggat ccttagagct agtgtacttg gtaactgcct tagtgccctc ggacacagca
     2041 tgcttagcca gctccccagg cagcagcagg cgcacagccg tctgaatctc cctggaggtg
     2101 atggtcgagc gcttattgta gtgagccagg cgagaagcct cgcccgcgat gcgctcgaag
     2161 atgtcgttga cgaaggagtt catgatcccc atggccttgg atgagatgcc ggtgtcgggg
     2221 tggacctgct tcagaacctt gtacacatag atagaatagc tctccttgcg gctgcgctta
     2281 cgcttcttac catccttctt ctgcgcctta gtgatagcct tcttagaacc ctttttaggg
     2341 gctggagcag acttagaggg ttcaggcatg gtggcggtac cggtggatcc ttgctagcat
     2401 aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat tatcactgtt
     2461 attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga agttatgaat
     2521 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     2581 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     2641 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     2701 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     2761 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     2821 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     2881 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     2941 cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct
     3001 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     3061 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     3121 cctccccgcc tggaattcga gctcggtaca gcttatcgat accgtcgact tcgagcaact
     3181 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3241 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3301 atgtctggat cgtctagcat cgaagatccc ccgatctgag gaacccctag tgatggagtt
     3361 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg
     3421 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc
     3481 caaccccccc cccccccccc ctgcagccca gctgcattaa tgaatcggcc aacgcgcggg
     3541 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3601 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3661 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3721 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3781 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3841 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3901 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta
     3961 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4021 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4081 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4141 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     4201 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4261 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4321 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4381 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4441 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4501 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4561 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4621 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4681 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4741 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4801 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4861 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4921 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4981 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5041 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5101 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5161 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5221 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5281 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5341 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5401 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5461 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     5521 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     5581 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     5641 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     5701 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
     5761 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aaacgttaat
     5821 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     5881 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     5941 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     6001 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     6061 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     6121 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     6181 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     6241 gcgccgctac agggcgcgtc gcgccattcg ccattcaggc tacgcaactg ttgggaaggg
     6301 cgatcggtgc gggcctcttc gctattacgc cagctggctg ca
//
