LOCUS       sequence-489096-        6740 bp    DNA     circular SYN 10-OCT-2025
DEFINITION  Fluorescent reporter gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6740)
  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 6740)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 10, 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-mCherry-WPRE-bGH
FEATURES             Location/Qualifiers
     source          1..6740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   23..152
                     /label=AAV2 ITR
     primer_bind     1014..1036
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1098..1117
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1190..1214
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1270..1647
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      1654..1663
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1660..2370
                     /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(1805..1823)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2073..2092
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2463..2487)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2531..2555)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2582..3170
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2635..2655)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3053..3064)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3197..3214)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3203..3427
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3637..3966)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3651..3786
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3951..3971
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4212..4800)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4292..4311)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4971..5762,5763..5831))
                     /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     5594..5613
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5832..5936)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6004..6022
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6060..6082)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6182..6201
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6415..6434
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6724..6740,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6724..6740,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaatacacgg aattaattct agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa
       61 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
      121 gggagtggcc aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta
      181 cttatctacg tagccatgct ctaggtacca ttgacgtcaa taatgacgta tgttcccata
      241 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      301 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      361 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      421 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      481 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      541 ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccrgg gggggggggg
      601 gggggggggg gggggggggg gggggggsgg rgrggkgsgg cggcagccaa tcagagcggc
      661 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
      721 agcgcgcggc gggcgggrgt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc
      781 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
      841 gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt
      901 ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc
      961 tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg
     1021 cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt
     1081 tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga
     1141 attggatctt ccctagaact agtggatccc ccgggctgca ggaattatca caagtttgta
     1201 caaaaaagca ggctccacca tgggaaccaa ttcagtcgcg tttagtgaac cgtcagaggt
     1261 accgccacca tgccagagcc agcgaagtct gctcccgccc cgaaaaaggg ctccaagaag
     1321 gcggtgacta aggcgcagaa gaaaggcggc aagaagcgca agcgcagccg caaggagagc
     1381 tattccatct atgtgtacaa ggttctgaag caggtccacc ctgacaccgg catttcgtcc
     1441 aaggccatgg gcatcatgaa ttcgtttgtg aacgacattt tcgagcgcat cgcaggtgag
     1501 gcttcccgcc tggcgcatta caacaagcgc tcgaccatca cctccaggga gatccagacg
     1561 gccgtgcgcc tgctgctgcc tggggagttg gccaagcacg ccgtgtccga gggtactaag
     1621 gccatcacca agtacaccag cgctaaggat cccgccacca tggtgagcaa gggcgaggag
     1681 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     1741 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     1801 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     1861 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     1921 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     1981 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     2041 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     2101 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     2161 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     2221 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     2281 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     2341 accggcggca tggacgagct gtacaagtaa agcggccgcg tcgagagatg ggggaggcta
     2401 actgagctag cttagtcatg caccgggctg caggaattcg cggccgcact cgagatatct
     2461 agacccagct ttcttgtaca aagtggtgat aattcgatat caagcttatc gataccgtcg
     2521 agatatctag acccagcttt cttgtacaaa gtggtgataa ttcgatatca agcttatcga
     2581 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2641 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2701 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2761 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2821 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     2881 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     2941 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     3001 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     3061 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     3121 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     3181 tcgactcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3241 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3301 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3361 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3421 tctatggctt ctgaggcgga aagaaccagc tggggctcga ctagagcatg gctacgtaga
     3481 taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac
     3541 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     3601 gggcggcctc agtgagcgag cgagcgcgca gagctttttg caaaagccta ggcctccaaa
     3661 aaagcctcct cactacttct ggaatagctc agaggccgag gcggcctcgg cctctgcata
     3721 aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg
     3781 cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg
     3841 catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag
     3901 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg
     3961 acacacattc cacagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4021 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4081 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4141 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4201 ttgctggcgt ttttccatag gctccgcccc cctgacaagc atcacaaaaa tcgacgctca
     4261 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4321 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4381 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4441 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4501 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4561 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4621 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4681 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4741 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4801 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4861 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4921 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4981 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5041 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5101 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5161 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5221 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5281 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5341 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5401 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5461 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     5521 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5581 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5641 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5701 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5761 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5821 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5881 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5941 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     6001 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     6061 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     6121 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     6181 gcggcatcag agcagattgt actgagagtg caccattcga cgctctccct tatgcgactc
     6241 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     6301 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     6361 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     6421 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     6481 tccggcgtag aggatctggc tagcgatgac cctgctgatt ggttcgctga ccatttccgg
     6541 gtgcgggacg gcgttaccag aaactcagaa ggttcgtcca accaaaccga ctctgacggc
     6601 agtttacgag agagatgata gggtctgctt cagtaagcca gatgctacac aattaggctt
     6661 gtacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
     6721 acgatttagg tgacactata
//
