LOCUS       sequence-395052-        6750 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ratiometric D4H 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 6750)
  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 6750)
  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_D4H
FEATURES             Location/Qualifiers
     source          1..6750
                     /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    2735..2768
                     /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    2819..2852
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2877..3465
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2930..2950)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3348..3359)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3497..3973
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3696..3715)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4013..4153
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4013..4142
                     /label=AAV2 ITR
     rep_origin      4228..4683
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4315..4334)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4525..4546
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4700..4719)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4819..4841
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4879..4897)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4965..5069
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5070..5138,5139..5930)
                     /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(5288..5307)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6101..6689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6590..6609
                     /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 tcccaagcaa ggcctgtaca
      961 ctctcttgct tttattctta tatttcttgc attagcttca agaggtatta ctgttgaata
     1021 gtgagctgtt ttagattgat aatttccatc ccatgtttta tgagttaaaa cttcatttcc
     1081 ttctttgtca tatgaaactt 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 tagcataact tcgtataaag tatcctatac
     2761 gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaat
     2821 aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt atcgataatc
     2881 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     2941 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3001 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3061 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3121 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3181 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3241 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3301 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3361 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3421 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
     3481 tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
     3541 ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
     3601 gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg
     3661 gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc
     3721 agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc
     3781 tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
     3841 tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca
     3901 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
     3961 cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc
     4021 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4081 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4141 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4201 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4261 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     4321 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4381 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4441 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4501 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     4561 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     4621 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     4681 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     4741 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4801 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4861 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     4921 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4981 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5041 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5101 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5161 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5221 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5281 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5341 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5401 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5461 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5521 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5581 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5641 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5701 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5761 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5821 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5881 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5941 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6001 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6061 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6121 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6181 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6241 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6301 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6361 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6421 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6481 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6541 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6601 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6661 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6721 tggccttttg ctggcctttt gctcacatgt
//
