LOCUS       sequence-385751-        6495 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ratiometric D4H sensor for in vivo astrocyte-specific cholesterol 
            visualization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6495)
  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 6495)
  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-GfaABC1D-eGFP-2A-mCherry_D4H
FEATURES             Location/Qualifiers
     source          1..6495
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      666..675
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(672..674,675..677,678..1388)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(672..674,675..677,678..1388)
                     /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(717..738)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(978..997)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1325..1346
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1437..1490
                     /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             1506..2207
                     /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(1645..1663)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1913..1932
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2208..2224
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2208..2224
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    2622..3210
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2675..2695)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3093..3104)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3242..3718
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3441..3460)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3758..3898
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3758..3887
                     /label=AAV2 ITR
     rep_origin      3973..4428
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4060..4079)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4270..4291
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4445..4464)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4564..4586
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4624..4642)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4710..4814
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4815..4883,4884..5675)
                     /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"
     rep_origin      5846..6434
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6335..6354
                     /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 gcgtagatct aacatatcct ggtgtggagt aggggacgct
      181 gctctgacag aggctcgggg gcctgagctg gctctgtgag ctggggagga ggcagacagc
      241 caggccttgt ctgcaagcag acctggcagc attgggctgg ccgcccccca gggcctcctc
      301 ttcatgccca gtgaatgact caccttggca cagacacaat gttcggggtg ggcacagtgc
      361 ctgcttcccg ccgcacccca gcccccctca aatgccttcc gagaagccca ttgagcaggg
      421 ggcttgcatt gcaccccagc ctgacagcct ggcatcttgg gataaaagca gcacagcccc
      481 ctaggggctg cccttgctgt gtggcgccac cggcggtgga gaacaaggct ctattcagcc
      541 tgtgcccagg aaaggggatc aggggatgcc caggcatgga cagtgggtgg caggggggga
      601 gaggagggct gtctgcttcc cagaagtcca aggacacaaa tgggtgaggg gagagctcac
      661 cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
      721 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
      781 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
      841 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
      901 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
      961 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1021 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1081 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1141 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1201 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1261 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     1321 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1381 tgtacaagtc cggactcaga tctcgagctc aagcttcgaa ttctgcagtc gacctcgagg
     1441 gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct gaattcatgg
     1501 caaggagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
     1561 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     1621 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
     1681 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
     1741 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     1801 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     1861 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     1921 tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
     1981 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg
     2041 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca
     2101 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
     2161 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtct agaactagtg
     2221 gatccaaggg aaaaataaac ttagatcata gtggagccta tgttgcacag tttgaagtag
     2281 cctgggatga agtttcatat gacaaagaag gaaatgaagt tttaactcat aaaacatggg
     2341 atggaaatta tcaatctaaa acagctcact attcaacagt aatacctctt gaagctaatg
     2401 caagaaatat aagaataaaa gcaagagagt gtacaggcct tgcttgggaa tggtggagag
     2461 atgttataag tgaatatgat gttccattaa caaataatat aaatgtttca atatggggaa
     2521 caactttata ccctggatct agtattactt acaattaagc ggccgccacc gcggtggagc
     2581 tcaagcactc catgtaggct agcgatatca agcttatcga taatcaacct ctggattaca
     2641 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     2701 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     2761 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     2821 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     2881 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     2941 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3001 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga
     3061 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3121 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     3181 gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcta
     3241 cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
     3301 agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc
     3361 cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac
     3421 aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg
     3481 gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt
     3541 gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg
     3601 gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc
     3661 ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct
     3721 gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     3781 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3841 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     3901 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3961 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4021 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     4081 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4141 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4201 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4261 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     4321 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     4381 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4441 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4501 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4561 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4621 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4681 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4741 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4801 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4861 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4921 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4981 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5041 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5101 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5161 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5221 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5281 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5341 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5401 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5461 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5521 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5581 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5641 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5701 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5761 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5821 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5881 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5941 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6001 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6061 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6121 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6181 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6241 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6301 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6361 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6421 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6481 cttttgctca catgt
//
