LOCUS       sequence-396773-        6383 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV transfer plasmid encoding a circularized RNA barcode D under U6 
            promoter and a subcellular localization protein binder under hSyn 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6383)
  AUTHORS   Shi H, He Y, Zhou Y, Huang J, Maher K, Wang B, Tang Z, Luo S, Tan P,
            Wu M, Lin Z, Ren J, Thapa Y, Tang X, Chan KY, Deverman BE, Shen H, 
            Liu A, Liu J, Wang X
  TITLE     Spatial atlas of the mouse central nervous system at molecular 
            resolution.
  JOURNAL   Nature. 2023 Oct;622(7983):552-561. doi: 10.1038/s41586-023-06569-5.
            Epub 2023 Sep 27.
  PUBMED    37758947
REFERENCE   2  (bases 1 to 6383)
  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"; doi: 
            "10.1038/s41586-023-06569-5"; journalName: "Nature"; date: 
            "2023-10"; volume: "622"; issue: "7983"; pages: "552-561"
COMMENT     Sequence Label: pAAV-U6-racRNA-D
FEATURES             Location/Qualifiers
     source          1..6383
                     /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        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        786..1233
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1253..1262
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1262..1303
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1880..1906
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             1931..1984
                     /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             1991..2014
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2510..3098
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2563..2583)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2981..2992)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3130..3606
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3329..3348)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3646..3786
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3646..3775
                     /label=AAV2 ITR
     rep_origin      3861..4316
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3948..3967)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4158..4179
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4333..4352)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4452..4474
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4512..4530)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4598..4702
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4703..4771,4772..5563)
                     /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(4921..4940)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5734..6322
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6223..6242
                     /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 gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgtgctcg cttcggcagc acatatacta
      421 gtcgacgggc cgcactcgcc ggtcccaagc ccggataaaa tgggaggggg cgggaaaccg
      481 cctaaccatg ccgagtgcgg ccgcttgcca tgtgtatcgg tccgggagca gacgatatgg
      541 cgtcgctccc ggtccgatac tctgatgatg ggtcccccta gggacgggac gttcgatgtt
      601 ggggaaaatc cgctgctcaa gattctataa actgtgcggt ccttcaattg gggtcccatc
      661 attcatggca agtggccgcg gtcggcgtgg actgtagaac actgccaatg ccggtcccaa
      721 gcccggataa aagtggaggg tacagtccac gctttttttc tagactgcag agggccctgc
      781 gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc ctacctgacg
      841 accgaccccg acccactgga caagcaccca acccccattc cccaaattgc gcatccccta
      901 tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca
      961 ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa
     1021 ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca ccttggtcgc
     1081 gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat aggggggcac
     1141 gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc
     1201 agcggaggag tcgtgtcgtg cctgagagcg cagtcgagaa ggtaccggat ccgccaccat
     1261 gggcaagccc atccccaacc ccctgctggg cctggacagc accggcggtg gaggttcctc
     1321 caaaaccatc gttctttcgg tcggcgaggc tactcgcact ctgactgaga tccagtccac
     1381 cgcagaccgt cagatcttcg aagagaaggt cgggcctctg gtgggtcggc tgcgcctcac
     1441 ggcttcgctc cgtcaaaacg gagccaagac cgcgtatcgc gtcaacctaa aactggatca
     1501 ggcggacgtc gttgattgct ccaccagcgt ctgcggcgag cttccgaaag tgcgctacac
     1561 tcaggtatgg tcgcacgacg tgacaatcgt tgcgaatagc accgaggcct cgcgcaaatc
     1621 gttgtacgat ttgaccaagt ccctcgtcgc gacctcgcag gtcgaagatc ttgtcgtcaa
     1681 ccttgtgccg ctgggccgtg gcggtggcgg atctggcggc ggtggtagca atgattttgg
     1741 caattacaac aatcagtctt ccaattttgg gccgatgaag ggaggaaact ttggaggcag
     1801 gagctctggc ccttatggtg gtggaggcca gtactttgct aaaccacgga accaaggtgg
     1861 ctatggcgga ggtggttctc tgcctccact tgaaagactg acactgggct caggaggatc
     1921 tggtggttct gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg
     1981 ccctgccatg gattacaagg atgacgacga taagggcgga ggtggttctt ccaaaaccat
     2041 cgttctttcg gtcggcgagg ctactcgcac tctgactgag atccagtcca ccgcagaccg
     2101 tcagatcttc gaagagaagg tcgggcctct ggtgggtcgg ctgcgcctca cggcttcgct
     2161 ccgtcaaaac ggagccaaga ccgcgtatcg cgtcaaccta aaactggatc aggcggacgt
     2221 cgttgattgc tccaccagcg tctgcggcga gcttccgaaa gtgcgctaca ctcaggtatg
     2281 gtcgcacgac gtgacaatcg ttgcgaatag caccgaggcc tcgcgcaaat cgttgtacga
     2341 tttgaccaag tccctcgtcg cgacctcgca ggtcgaagat cttgtcgtca accttgtgcc
     2401 gctgggccgt ggcggaggtg gttctaagct gaaccctcct gatgagagtg gccccggctg
     2461 catgagctgc tgtgtgctct cctaagaatt cgatatcaag cttatcgata atcaacctct
     2521 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     2581 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     2641 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     2701 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     2761 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     2821 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     2881 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc
     2941 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     3001 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     3061 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga
     3121 gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
     3181 gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
     3241 taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
     3301 gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
     3361 caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
     3421 cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
     3481 tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
     3541 tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
     3601 gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga
     3661 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     3721 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     3781 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3841 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3901 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     3961 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4021 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     4081 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4141 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     4201 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     4261 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     4321 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     4381 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4441 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4501 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     4561 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4621 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4681 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4741 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4801 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4861 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4921 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4981 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5041 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5101 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5161 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5221 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     5281 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5341 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5401 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5461 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5521 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5581 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5641 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5701 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5761 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5821 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5881 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5941 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6001 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6061 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6121 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6181 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6241 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6301 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6361 ttgctggcct tttgctcaca tgt
//
