LOCUS       sequence-345003-        5980 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid used for Crispr/cas9 based disruption of mouse Trim28, guide
            targeting exon 3. Expressing nuclear RFP. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5980)
  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 5980)
  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.U6gT28.3.hSyn.H2B.RFP
FEATURES             Location/Qualifiers
     source          1..5980
                     /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(3393..3981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3473..3492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4152..4943,4944..5012))
                     /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     4775..4794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5013..5117)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5185..5203
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5241..5263)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5363..5382
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5449..5904)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5586..5607)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5798..5817
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gggggggggg ggkggrsbbs svccmcymss kskctscscg ctcgctcgct 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 acaccggcac tccacacaat agctaggttt 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 ttwtcatgtc tggatcgtct agcatcgaag atcccccgat ctgaggaacc
     3121 cctagtgatg gagttggcca ctcccycycn tgcagcctgc attaatgaat cggccaacgc
     3181 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3241 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3301 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3361 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3421 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3481 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3541 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3601 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3661 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3721 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3781 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3841 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3901 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3961 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4021 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4081 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4141 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4201 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4261 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4321 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4381 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4441 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4501 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4561 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4621 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4681 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4741 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4801 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4861 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4921 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4981 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5041 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5101 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     5161 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     5221 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     5281 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     5341 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     5401 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg
     5461 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     5521 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     5581 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     5641 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     5701 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     5761 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     5821 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     5881 ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg
     5941 gcgatcggtg cgggcctctt cgctattacg ccaggctgca
//
