LOCUS       sequence-436323-        6124 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C terminal fusion of EGFP to mouse HP1-gamma (Cbx3) containing W174A
            mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6124)
  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 6124)
  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-gamma-W174A-EGFP
FEATURES             Location/Qualifiers
     source          1..6124
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          682..1698
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1622..1644
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1706..1725
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2333..2342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2339..2341,2342..2344,2345..3058)
                     /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(2339..2341,2342..2344,2345..3055)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2384..2405)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2645..2664)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2992..3013
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3145..3164)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3210..3265
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3264..3283)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3626..3642)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3626..3642)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3639..3661)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3650..3666
                     /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(3674..3680,3681..3698,3699..3704))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3719..3740
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3799..3994
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3845..3980
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4000..4134
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4037..4056)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4091..4110
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4202..4219)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4373..4961)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4453..4472)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5132..5923,5924..5992))
                     /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     5755..5774
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5993..6097)
                     /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 ggggggggtc ggctggggtg gggggcgcgg gggggggggg
      541 gggggggggg gggggggggg ggggggggcg aggcggaggg gtgcggcggc agccaatcag
      601 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
      661 aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc
      721 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc
      781 gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt
      841 tcttttctgt ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg
      901 agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg
      961 ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg
     1021 cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac
     1081 aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc
     1141 gggctgcaac cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt
     1201 gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag
     1261 gtgggggtgc cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc
     1321 ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta
     1381 tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa
     1441 tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc
     1501 aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct
     1561 ctccagcctc ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg
     1621 gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc
     1681 ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt
     1741 ggcaaagaat tctgcagtcg acggtaccta aaaaatggcc tccaataaaa ctacattgca
     1801 aaaaatggga aagaaacaaa atggaaagag taaaaaagtt gaagaggcag agcctgaaga
     1861 atttgtggta gaaaaagtac tggaccgtcg tgtagtgaat gggaaggtgg agtatttcct
     1921 gaagtggaag gggttcacag atgctgataa tacttgggaa ccagaagaaa atttagattg
     1981 tccagaatta attgaagcat ttcttaattc tcaaaaagct ggtaaagaaa aagatggtac
     2041 aaaaaggaaa tctttatctg acagtgaatc tgatgatagc aaatcgaaga agaagagaga
     2101 tgctgctgac aaaccaaggg gctttgccag aggtctcgac cctgaacgaa taatcggcgc
     2161 cacagacagc agcggagagt taatgtttct catgaagtgg aaggactcgg acgaggccga
     2221 cttggtgctg gcaaaggagg cgaacatgaa gtgtcctcag attgtcattg ccttctacga
     2281 ggagcggctg actgcgcatt cttgtcctga agatgaagca caattaccgg tcgccaccat
     2341 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     2401 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     2461 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     2521 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     2581 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     2641 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     2701 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2761 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2821 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2881 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2941 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     3001 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag
     3061 cggccgcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc
     3121 cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat
     3181 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
     3241 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
     3301 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat
     3361 gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc
     3421 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt
     3481 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct
     3541 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct
     3601 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3661 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3721 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3781 tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3841 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3901 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     3961 aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat
     4021 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4081 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc
     4141 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4201 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4261 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4321 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4381 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4441 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4501 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4561 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4621 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4681 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4741 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4801 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4861 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4921 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4981 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5041 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5101 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5161 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5221 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5281 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5341 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5401 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5461 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5521 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5581 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5641 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5701 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5761 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5821 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5881 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5941 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6001 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6061 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6121 ctgg
//
