LOCUS       sequence-345004-        5977 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 5977)
  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 5977)
  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..5977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     356..375
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        455..530
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        570..1017
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1060..1437
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      1450..1459
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1456..2166
                     /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(1601..1619)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1869..1888
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2185..2773
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2238..2258)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2656..2667)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2821..2955
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2858..2877)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2912..2931
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3390..3978)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3470..3489)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4149..4940,4941..5009))
                     /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     4772..4791
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5010..5114)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5182..5200
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5238..5260)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5360..5379
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5446..5901)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5583..5604)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5795..5814
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gggggggggg ggggrrsdss mcmcyccckc kmkgcscssy cgctcgctca ctgaggccgc
       61 ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
      121 gcgcagagag ggagtggcca actccatcac taggggttcc tcagatcggg ctgcaggaat
      181 tggccgctct agatttccca tgattccttc atatttgcat atacgataca aggctgttag
      241 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
      301 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
      361 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
      421 aggacgaaac accgcgcact ggttacacat gaccgtttta gagctagaaa tagcaagtta
      481 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttcta
      541 gactctagct gcagagggac ctgcgtatga gtgcaagtgg gttttaggac caggatgagg
      601 cggggtgggg gtgcctacct gacgaccgac cccgacccac tggacaagca cccaaccccc
      661 attccccaaa ttgcgcatcc cctatcagag agggggaggg gaaacaggat gcggcgaggc
      721 gcgtgcgcac tgccagcttc agcaccgcgg acagtgcctt cgcccccgcc tggcggcgcg
      781 cgccaccgcc gcctcagcac tgaaggcgcg ctgacgtcac tcgccggtcc cccgcaaact
      841 ccccttcccg gccaccttgg tcgcgtccgc gccgccgccg gcccagccgg accgcaccac
      901 gcgaggcgcg agataggggg gcacgggcgc gaccatctgc gctgcggcgc cggcgactca
      961 gcgctgcctc agtctgcggt gggcagcgga ggagtcgtgt cgtgcctgag agcgcagtcg
     1021 aattcaagct gctagcaagg atccaccggt accgccacca tgcctgaacc ctctaagtct
     1081 gctccagccc ctaaaaaggg ttctaagaag gctatcacta aggcgcagaa gaaggatggt
     1141 aagaagcgta agcgcagccg caaggagagc tattctatct atgtgtacaa ggttctgaag
     1201 caggtccacc ccgacaccgg catctcatcc aaggccatgg ggatcatgaa ctccttcgtc
     1261 aacgacatct tcgagcgcat cgcgggcgag gcttctcgcc tggctcacta caataagcgc
     1321 tcgaccatca cctccaggga gattcagacg gctgtgcgcc tgctgctgcc tggggagctg
     1381 gctaagcatg ctgtgtccga gggcactaag gcagttacca agtacactag ctctaaggat
     1441 ccaccggtcg ccaccatggt gagcaagggc gaggaggata acatggccat catcaaggag
     1501 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
     1561 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     1621 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
     1681 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
     1741 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     1801 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     1861 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg
     1921 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
     1981 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     2041 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
     2101 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     2161 aagtaaacta gtggatccgt cgacaatcaa cctctggatt acaaaatttg tgaaagattg
     2221 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     2281 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     2341 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     2401 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     2461 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     2521 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag
     2581 ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     2641 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     2701 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     2761 gccgcctccc cgcctggaat tcgagctcgg tacagcttat cgataccgtc gacttcgagc
     2821 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2881 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     2941 tatcatgtct ggatcgtcta gcatcgaaga tcccccgatc tgaggaaccc ctagtgatgg
     3001 agttggccac tccctctctg cdsscyssmw ydmtvrwtsd gsynnykskc srrrmkmrkc
     3061 rrkktrystw wtsrkstctg satcgtctag catcgaagat cccccgatct gaggaacccc
     3121 tagtgatgga gttggccact cccycyctgc agcctgcatt aatgaatcgg ccaacgcgcg
     3181 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3241 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3301 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3361 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3421 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3481 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3541 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3601 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3661 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3721 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3781 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3841 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3901 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3961 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4021 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4081 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4141 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4201 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4261 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4321 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4381 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4441 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4501 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4561 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4621 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4681 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4741 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4801 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4861 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4921 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4981 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5041 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5101 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     5161 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     5221 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     5281 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     5341 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     5401 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta
     5461 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     5521 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     5581 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     5641 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     5701 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     5761 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     5821 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     5881 atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg
     5941 atcggtgcgg gcctcttcgc tattacgcca ggctgca
//
