LOCUS       sequence-345058-        6333 bp    DNA     circular SYN 15-APR-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 6333)
  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 6333)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..6333
                     /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(3746..4334)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3826..3845)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4505..5296,5297..5365))
                     /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     5128..5147
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5366..5470)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5538..5556
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5594..5616)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5716..5735
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5802..6257)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5939..5960)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6151..6170
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gggggggggg gggkgrsbvv vyccmyymss kvksykyscs 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 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 tctctgcdss cyssmwydmt vrhtsdgsyn nykskcsrrr mkmrkcrrtk
     3421 trycttmtsr tgtctggatc gtctagcatc gaagatcccc cgatctgagg aacccctagt
     3481 gatggagttg gccactcccy cyctgcagcc tgcattaatg aatcggccaa cgcgcgggga
     3541 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3601 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3661 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3721 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3781 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3841 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3901 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3961 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4021 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4081 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4141 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4201 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4261 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4321 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4381 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4441 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4501 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4561 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4621 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4681 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4741 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4801 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4861 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4921 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4981 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5041 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5101 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5161 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5221 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5281 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5341 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5401 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5461 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     5521 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     5581 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     5641 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     5701 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     5761 aaataccgca cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat
     5821 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     5881 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     5941 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     6001 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     6061 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     6121 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     6181 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     6241 gccgctacag ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     6301 gtgcgggcct cttcgctatt acgccaggct gca
//
