LOCUS       sequence-385790-        6479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ratiometric D4H sensor for in vivo neuron-specific cholesterol 
            visualization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6479)
  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 6479)
  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-eGFP-2A-mCherry_D4H
FEATURES             Location/Qualifiers
     source          1..6479
                     /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)"
     regulatory      650..659
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(656..658,659..661,662..1372)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(656..658,659..661,662..1372)
                     /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(701..722)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(962..981)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1309..1330
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1421..1474
                     /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             1490..2191
                     /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(1629..1647)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1897..1916
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2192..2208
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2192..2208
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    2606..3194
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2659..2679)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3077..3088)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3226..3702
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3425..3444)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3742..3882
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3742..3871
                     /label=AAV2 ITR
     rep_origin      3957..4412
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4044..4063)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4254..4275
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4429..4448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4548..4570
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4608..4626)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4694..4798
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4799..4867,4868..5659)
                     /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(5017..5036)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5830..6418
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6319..6338
                     /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 gtaccggacg ctaccggtcg ccaccatggt
      661 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      721 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      781 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      841 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
      901 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
      961 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1021 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1081 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1141 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1201 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1261 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1321 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact
     1381 cagatctcga gctcaagctt cgaattctgc agtcgacctc gagggcagag gaagtcttct
     1441 aacatgcggt gacgtggagg agaatcccgg ccctgaattc atggcaagga gcaagggcga
     1501 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     1561 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     1621 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     1681 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     1741 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     1801 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     1861 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     1921 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     1981 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     2041 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     2101 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     2161 ctccaccggc ggcatggacg agctgtacaa gtctagaact agtggatcca agggaaaaat
     2221 aaacttagat catagtggag cctatgttgc acagtttgaa gtagcctggg atgaagtttc
     2281 atatgacaaa gaaggaaatg aagttttaac tcataaaaca tgggatggaa attatcaatc
     2341 taaaacagct cactattcaa cagtaatacc tcttgaagct aatgcaagaa atataagaat
     2401 aaaagcaaga gagtgtacag gccttgcttg ggaatggtgg agagatgtta taagtgaata
     2461 tgatgttcca ttaacaaata atataaatgt ttcaatatgg ggaacaactt tataccctgg
     2521 atctagtatt acttacaatt aagcggccgc caccgcggtg gagctcaagc actccatgta
     2581 ggctagcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     2641 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     2701 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     2761 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     2821 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     2881 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     2941 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     3001 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc
     3061 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     3121 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     3181 ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt
     3241 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     3301 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     3361 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     3421 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     3481 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     3541 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     3601 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     3661 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     3721 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     3781 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     3841 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     3901 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     3961 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4021 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4081 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4141 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     4201 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4261 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     4321 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4381 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     4441 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     4501 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4561 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     4621 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4681 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4741 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4801 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4861 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4921 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4981 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5041 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5101 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5161 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5221 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5281 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5341 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5401 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5461 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5521 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5581 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5641 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5701 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5761 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5821 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5881 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5941 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6001 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6061 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6121 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6181 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6241 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6301 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6361 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6421 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
