LOCUS       sequence-345059-        5979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid used for Crispr/cas9 based disruption of mouse Trim28, guide
            targeting exon 4. Expressing nuclear RFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5979)
  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 5979)
  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.H2B.RFP
FEATURES             Location/Qualifiers
     source          1..5979
                     /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             1062..1439
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      1452..1461
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1458..2168
                     /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(1603..1621)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1871..1890
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2187..2775
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2240..2260)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2658..2669)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2823..2957
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2860..2879)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2914..2933
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3392..3980)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3472..3491)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4151..4942,4943..5011))
                     /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     4774..4793
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5012..5116)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5184..5202
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5240..5262)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5362..5381
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5448..5903)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5585..5606)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5797..5816
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gggggggggg ggggggsvss myscmnyssc kskcygcscg cycgctcgct cactgaggcc
       61 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc 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 gccggcgact
      961 cagcgctgcc tcagtctgcg gtgggcagcg gaggagtcgt gtcgtgcctg agagcgcagt
     1021 cgaattcaag ctgctagcaa ggatccaccg gtaccgccac catgcctgaa ccctctaagt
     1081 ctgctccagc ccctaaaaag ggttctaaga aggctatcac taaggcgcag aagaaggatg
     1141 gtaagaagcg taagcgcagc cgcaaggaga gctattctat ctatgtgtac aaggttctga
     1201 agcaggtcca ccccgacacc ggcatctcat ccaaggccat ggggatcatg aactccttcg
     1261 tcaacgacat cttcgagcgc atcgcgggcg aggcttctcg cctggctcac tacaataagc
     1321 gctcgaccat cacctccagg gagattcaga cggctgtgcg cctgctgctg cctggggagc
     1381 tggctaagca tgctgtgtcc gagggcacta aggcagttac caagtacact agctctaagg
     1441 atccaccggt cgccaccatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg
     1501 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
     1561 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     1621 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
     1681 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     1741 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     1801 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     1861 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
     1921 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
     1981 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
     2041 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca
     2101 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt
     2161 acaagtaaac tagtggatcc gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     2221 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     2281 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     2341 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     2401 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     2461 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     2521 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     2581 agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     2641 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     2701 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     2761 gggccgcctc cccgcctgga attcgagctc ggtacagctt atcgataccg tcgacttcga
     2821 gcaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     2881 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     2941 cttatcatgt ctggatcgtc tagcatcgaa gatcccccga tctgaggaac ccctagtgat
     3001 ggagttggcc actccctctc tgcdsscyss mwywmtrrwt skgsynnyks kcsrrrmkmr
     3061 kcrrtktryc twwtsrtgtc tggatcgtct agcatcgaag atcccccgat ctgaggaacc
     3121 cctagtgatg gagttggcca ctcccycyct gcagcctgca ttaatgaatc ggccaacgcg
     3181 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3241 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3301 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3361 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3421 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3481 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3541 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3601 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3661 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3721 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3781 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3841 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3901 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3961 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4021 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4081 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4141 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4201 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4261 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4321 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4381 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4441 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4501 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4561 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4621 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4681 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4741 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4801 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4861 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4921 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4981 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5041 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5101 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     5161 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     5221 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     5281 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     5341 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     5401 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt
     5461 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     5521 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     5581 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     5641 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     5701 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     5761 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     5821 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     5881 taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg
     5941 cgatcggtgc gggcctcttc gctattacgc caggctgca
//
