LOCUS       sequence-385744-        6751 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ratiometric YDA sensor for in vivo cre-dependent neuron-specific 
            cholesterol visualization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6751)
  AUTHORS   Czernecki C, Dixit S, Riezman I, Innocenti S, Bornmann C, Pfrieger 
            FW, Riezman H, Scheiffele P
  TITLE     Cell type-specific assessment of cholesterol distribution in models 
            of neurodevelopmental disorders
  JOURNAL   bioRxiv 2022.11.16.516849
REFERENCE   2  (bases 1 to 6751)
  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: "bioRxiv 
            2022.11.16.516849"
COMMENT     Sequence Label: pAAV-Syn-DiO-eGFP-2A-mCherry_YDA
FEATURES             Location/Qualifiers
     source          1..6751
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /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(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1166..1182)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1166..1182)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             complement(1183..1884)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="SKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQ
                     TAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFED
                     GGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGE
                     IKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG
                     RHSTGGMDELYK"
     primer_bind     complement(1458..1477)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1727..1745
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1900..1953)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(join(2002..2712,2713..2715,2716..2718))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2002..2712,2713..2715,2716..2718))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2044..2065)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2393..2412
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2652..2673
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2715..2724
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2736..2769
                     /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    2820..2853
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2878..3466
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2931..2951)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3349..3360)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3498..3974
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3697..3716)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4014..4154
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4014..4143
                     /label=AAV2 ITR
     rep_origin      4229..4684
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4316..4335)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4526..4547
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4701..4720)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4820..4842
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4880..4898)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4966..5070
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5071..5139,5140..5931)
                     /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(5289..5308)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6102..6690
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6591..6610
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgatcgcta gcctacatgg agtgcttgag ctccaccgcg gtggcggccg cttaattgta
      841 agtaatacta gatccagggt ataaagttgt tccccatatt gaaacattta tattatttgt
      901 taatggaaca tcatattcac ttataacatc tctccaccat tcccaccaaa ggcctgtaca
      961 ctctcttgct tttattctta tatttcttgc attagcttca agaggtatta ctgttgaata
     1021 gtgagctgtt ttccattgat aatttccatc ccatgtttta tgagttaaaa cttcatttcc
     1081 ttctttgtca gctgaaactt catcccaggc tacttcaaac tgtgcaacat aggctccact
     1141 atgatctaag tttatttttc ccttggatcc actagttcta gacttgtaca gctcgtccat
     1201 gccgccggtg gagtggcggc cctcggcgcg ttcgtactgt tccacgatgg tgtagtcctc
     1261 gttgtgggag gtgatgtcca acttgatgtt gacgttgtag gcgccgggca gctgcacggg
     1321 cttcttggcc ttgtaggtgg tcttgacctc agcgtcgtag tggccgccgt ccttcagctt
     1381 cagcctctgc ttgatctcgc ccttcagggc gccgtcctcg gggtacatcc gctcggagga
     1441 ggcctcccag cccatggtct tcttctgcat tacggggccg tcggagggga agttggtgcc
     1501 gcgcagcttc accttgtaga tgaactcgcc gtcctgcagg gaggagtcct gggtcacggt
     1561 caccacgccg ccgtcctcga agttcatcac gcgctcccac ttgaagccct cggggaagga
     1621 cagcttcaag tagtcgggga tgtcggcggg gtgcttcacg taggccttgg agccgtacat
     1681 gaactgaggg gacaggatgt cccaggcgaa gggcaggggg ccacccttgg tcaccttcag
     1741 cttggcggtc tgggtgccct cgtaggggcg gccctcgccc tcgccctcga tctcgaactc
     1801 gtggccgttc acggagccct ccatgtgcac cttgaagcgc atgaactcct tgatgatggc
     1861 catgttatcc tcctcgccct tgctccttgc catgaattca gggccgggat tctcctccac
     1921 gtcaccgcat gttagaagac ttcctctgcc ctcgaggtcg actgcagaat tcgaagcttg
     1981 agctcgagat ctgagtccgg acttgtacag ctcgtccatg ccgagagtga tcccggcggc
     2041 ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc
     2101 ggactgggtg ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt
     2161 gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt
     2221 gaagttcacc ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt
     2281 gtagttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt
     2341 cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta
     2401 gttgccgtcg tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga
     2461 cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta
     2521 ggtcagggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa
     2581 cttcagggtc agcttgccgt aggtggcatc gccctcgccc tcgccggaca cgctgaactt
     2641 gtggccgttt acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc
     2701 ctcgcccttg ctcaccatgg tggcgaccgg ctagcataac ttcgtataaa gtatcctata
     2761 cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa
     2821 taacttcgta taatgtatgc tatacgaagt tatgaattcg atatcaagct tatcgataat
     2881 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     2941 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3001 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3061 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3121 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3181 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3241 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3301 tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3361 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3421 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg
     3481 ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc
     3541 tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt
     3601 tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg
     3661 ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg
     3721 cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc
     3781 ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt
     3841 ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc
     3901 aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc
     3961 ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc
     4021 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4081 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     4141 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4201 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     4261 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     4321 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4381 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4441 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4501 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4561 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     4621 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     4681 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     4741 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4801 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4861 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     4921 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4981 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5041 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5101 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5161 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5221 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5281 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5341 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5401 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5461 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5521 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5581 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5641 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5701 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5761 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5821 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5881 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5941 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6001 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6061 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6121 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6181 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6241 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6301 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6361 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6421 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6481 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6541 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6601 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6661 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6721 ctggcctttt gctggccttt tgctcacatg t
//
