LOCUS       sequence-489036-        5126 bp    DNA     circular SYN 09-OCT-2025
DEFINITION  Fluorescent reporter gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5126)
  AUTHORS   Rybarikova M, Rey M, Hasanovic E, Sipion M, Rambousek L, Deglon N
  TITLE     Gene editing for Spinocerebellar ataxia type 3 taking advantage of 
            the human ATXN3L paralog as replacement gene.
  JOURNAL   Gene Ther. 2025 Jul 28. doi: 10.1038/s41434-025-00557-2.
  PUBMED    40721863
REFERENCE   2  (bases 1 to 5126)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 9, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Gene Ther. 
            2025 Jul 28. doi: 10.1038/s41434-025-00557-2."
COMMENT     Sequence Label: pAAVss-CBA-H2B-staygold-SYNpA
FEATURES             Location/Qualifiers
     source          1..5126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    819..843
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(862..886)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        1163..1440
                     /label=chicken beta-actin promoter
     primer_bind     1719..1741
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1803..1822
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1855..2232
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      2245..2254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2251..2901
                     /codon_start=1
                     /product="bright and highly photostable dimeric green 
                     fluorescent protein (Hirano et al., 2022)"
                     /label=StayGold
                     /note="engineered variant of a GFP from Cytaeis uchidae"
                     /translation="MASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKL
                     PMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFM
                     KNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNEVQHIPRDDGVECPVTLLYPLLSDKS
                     KCVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
     polyA_signal    2983..3031
                     /note="synthetic polyadenylation signal"
     repeat_region   3039..3179
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3039..3168
                     /label=AAV2 ITR
     rep_origin      3254..3709
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3341..3360)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3551..3572
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3726..3745)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3845..3867
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3905..3923)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3991..4095
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4096..4164,4165..4956)
                     /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     complement(4314..4333)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggcctcta gtggatcccc cgggctgcag gaattatcac aagtttgtac aaaaaagcag
      841 gcttcatcgc tagtaggcct gacccagctt tcttgtacaa agtggtgata attcgatatc
      901 aagcttgcat gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      961 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     1021 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     1081 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     1141 ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc
     1201 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
     1261 atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
     1321 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
     1381 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
     1441 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
     1501 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
     1561 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
     1621 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gctgtccgcg
     1681 gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac
     1741 cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct
     1801 gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattgg atccatgcca
     1861 gagccagcga agtctgctcc cgccccgaaa aagggctcca agaaggcggt gactaaggcg
     1921 cagaagaaag gcggcaagaa gcgcaagcgc agccgcaagg agagctattc catctatgtg
     1981 tacaaggttc tgaagcaggt ccaccctgac accggcattt cgtccaaggc catgggcatc
     2041 atgaattcgt ttgtgaacga cattttcgag cgcatcgcag gtgaggcttc ccgcctggcg
     2101 cattacaaca agcgctcgac catcacctcc agggagatcc agacggccgt gcgcctgctg
     2161 ctgcctgggg agttggccaa gcacgccgtg tccgagggta ctaaggccat caccaagtac
     2221 accagcgcta aggatccccc ggtcgccacc atggccagca cccccttcaa gttccagctg
     2281 aagggcacca tcaacggcaa gagcttcacc gtggaaggcg agggcgaggg caatagccac
     2341 gagggcagcc acaaaggcaa gtacgtgtgc accagcggca aactgccaat gtcttgggcc
     2401 gccctgggaa ctagcttcgg ctatggcatg aagtactaca ccaagtaccc cagcggcctg
     2461 aagaactggt tccacgaggt gatgcccgag ggcttcacct acgacagaca catccagtac
     2521 aagggcgacg gcagcatcca cgccaagcac cagcacttca tgaagaacgg cacctaccac
     2581 aacatcgtgg agttcaccgg ccaggacttc aaggagaaca gccccgtgct gaccggcgac
     2641 atgaacgtga gcctgcccaa cgaggtgcag cacatcccca gagatgacgg cgtggagtgc
     2701 ccagtgaccc tgctgtaccc tctgctgagc gacaagagca agtgcgtgga ggcccaccag
     2761 aacaccatct gcaagcccct gcacaatcag ccagcccccg atgtgccata ccactggatc
     2821 agaaagcagt acacccagag caaggacgac accgaggaga gagaccacat ctgccagagc
     2881 gagaccctgg aggcccacct gtaagatatc acctcgcgac tagctgatca gcctcgagta
     2941 cctttaagac caatgactta caaggcagct gtagatcaat tcaataaaag atctttattt
     3001 tcattagatc tgtgtgttgg ttttttgtgt gcggccgcag gaacccctag tgatggagtt
     3061 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     3121 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     3181 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     3241 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3301 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     3361 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3421 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     3481 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3541 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     3601 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     3661 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     3721 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     3781 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3841 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     3901 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     3961 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4021 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4081 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4141 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4201 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4261 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4321 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4381 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4441 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     4501 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     4561 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     4621 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     4681 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4741 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4801 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4861 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4921 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4981 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5041 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5101 cccgtagaaa agatcaaagg atcttc
//
