LOCUS       sequence-436318-        6145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C terminal fusion of GFP to mouse HP1-alpha (Cbx5) containing I165E 
            mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6145)
  AUTHORS   Newman AG, Sharif J, Bessa P, Zaqout S, Brown JP, Richter D, 
            Dannenberg R, Nakayama M, Mueller S, Schaub T, Manickaraj S, 
            Boehm-Sturm P, Ohara O, Koseki H, Singh PB, Tarabykin V
  TITLE     Glial reactivity and cognitive decline follow chronic 
            heterochromatin loss in neurons.
  JOURNAL   Nat Commun. 2025 Aug 8;16(1):7325. doi: 10.1038/s41467-025-61319-7.
  PUBMED    40781223
REFERENCE   2  (bases 1 to 6145)
  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: "Nat 
            Commun."; date: "2025-08-8"; volume: "16(1):7325. doi"; pages: " 
            10.1038/s41467-025-61319-7"
COMMENT     Sequence Label: pCAG-HP1-alpha-I165E-EGFP
FEATURES             Location/Qualifiers
     source          1..6145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          679..1695
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1619..1641
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1703..1722
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2354..2363
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2360..2362,2363..2365,2366..3079)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2360..2362,2363..2365,2366..3076)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2405..2426)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2666..2685)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3013..3034
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3166..3185)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3231..3286
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3285..3304)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3647..3663)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3647..3663)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3660..3682)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3671..3687
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3695..3701,3702..3719,3720..3725))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3740..3761
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3820..4015
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3866..4001
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4021..4155
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4058..4077)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4112..4131
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4223..4240)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4394..4982)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4474..4493)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5153..5944,5945..6013))
                     /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     5776..5795
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6014..6118)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggggcggg cccggggggg gggggggggg gggggggggg
      541 gggggggggg aggggcgggg gggggcgagg cggagaggtg cggcggcagc caatcagagc
      601 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
      661 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
      721 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
      781 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct
      841 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc
      901 ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg
      961 cccggcggct gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc
     1021 gaggggagcg cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa
     1081 ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg
     1141 ctgcaacccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg
     1201 gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg
     1261 ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc
     1321 ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg
     1381 taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct
     1441 gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg
     1501 aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc
     1561 cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg
     1621 ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc
     1681 ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc
     1741 aaagaattct gcagtcgacg gtacccacga catgggaaag aagaccaaga ggacagccga
     1801 cagctcttct tcagaggatg aggaggaata tgtggtggaa aaggtgttgg acaggcgcat
     1861 ggttaagggg caagtggaat atctgttgaa gtggaaaggc ttttctgagg agcacaatac
     1921 ttgggaacct gagaagaact tggattgtcc tgaactaatt tctgagttta tgaaaaagta
     1981 taagaagatg aaggagggtg aaaacaataa gcccagggag aaatcagaag gaaacaagag
     2041 gaaatccagt ttctccaaca gcgctgatga tattaaatct aaaaaaaaga gagagcaaag
     2101 caatgatatc gctcggggct ttgagagagg actggaacca gaaaagatca tcggagcaac
     2161 agattcctgc ggtgacttaa tgttcttaat gaaatggaaa gacacagatg aagctgacct
     2221 ggttcttgca aaagaagcta acgtgaagtg tccacagatt gtggaggcat tttatgaaga
     2281 gagactgacg tggcacgcat atccagagga tgcggaaaac aaagaaaaag aaagcgcgaa
     2341 gagcttaccg gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     2401 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     2461 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     2521 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     2581 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     2641 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     2701 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     2761 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     2821 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     2881 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     2941 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     3001 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     3061 ggacgagctg tacaagtaaa gcggccgcac tcctcaggtg caggctgcct atcagaaggt
     3121 ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt ttccctctgc
     3181 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     3241 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata
     3301 tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata
     3361 tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa
     3421 acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt
     3481 ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt
     3541 tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct
     3601 cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc
     3661 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3721 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3781 ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt agtcagcaac
     3841 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3901 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     3961 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     4021 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4081 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4141 tatcatgtct ggatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4201 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4261 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4321 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4381 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4441 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4501 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4561 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4621 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4681 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4741 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4801 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4861 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4921 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4981 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5041 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5101 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5161 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5221 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5281 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5341 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5401 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5461 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5521 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5581 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5641 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5701 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5761 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5821 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5881 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5941 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6001 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6061 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6121 catttccccg aaaagtgcca cctgg
//
