LOCUS       sequence-361683-        6269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid is for use with neuronal cell-type selective activity 
            tagging. The Caspase3 expression requires both neural activity and 
            Cre recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6269)
  AUTHORS   Yu X, Zhao G, Wang D, Wang S, Li R, Li A, Wang H, Nollet M, Chun YY,
            Zhao T, Yustos R, Li H, Zhao J, Li J, Cai M, Vyssotski AL, Li Y, 
            Dong H, Franks NP, Wisden W
  TITLE     A specific circuit in the midbrain detects stress and induces 
            restorative sleep.
  JOURNAL   Science. 2022 Jul;377(6601):63-72. doi: 10.1126/science.abn0853. 
            Epub 2022 Jun 30.
  PUBMED    35771921
REFERENCE   2  (bases 1 to 6269)
  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: 
            "10.1126/science.abn0853"; journalName: "Science"; date: "2022-07"; 
            volume: "377"; issue: "6601"; pages: "63-72"
COMMENT     Sequence Label: pAAV-TRE-DIO-Casp3-TEV
FEATURES             Location/Qualifiers
     source          1..6269
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    36..54
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    71..89
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    107..125
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    143..161
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    178..196
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    214..232
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        244..282
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     279..303
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(346..379)
                     /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(430..463)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1180..1233)
                     /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(1549..1569)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     regulatory      2088..2097
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2105..2138
                     /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    2189..2222
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2247..2835
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2300..2320)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2718..2729)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2867..3343
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3066..3085)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3383..3523
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3383..3512
                     /label=AAV2 ITR
     rep_origin      3598..4053
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3685..3704)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3895..3916
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4070..4089)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4189..4211
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4249..4267)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4335..4439
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4440..4508,4509..5300)
                     /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(4658..4677)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5471..6059
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5960..5979
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6121..6250
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    join(6269,1..18)
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgatg
       61 tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat
      121 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttatcc
      181 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt
      241 cgaggtaggc gtgtacggtg ggaggcctat ataagcagag ctcgtttagt gaaccgtcag
      301 atcgcctgga gaattcgagc tcggtacccg ggggtcgact ccggaataac ttcgtatagg
      361 atactttata cgaagttatg cagaatggta gctggattgt agctgctatt agcaatatga
      421 aacctcttaa taacttcgta tagcatacat tatacgaagt tatggcgcgc cttctcaaag
      481 ctgagtggct tccttaacgg gctggaaggg ttcctcgggc ttgaccatga acaccttatg
      541 tccgccccaa agaacgctgt cggcgttcag cctccagccg ctcacccact gctgcgcctc
      601 ttgattggtc aacagctcca tgaagttttt cggcacgctc gtgaagtagt tgttggtatt
      661 ggtaaaattg ctggcgctgt ggatgcccac gatgaagccg tccctagtgc tcaccaatgg
      721 gctgccgcat tggccatcct tcgtctgaat ccagtgcttc cagaagatgc cgtcgctgct
      781 ggggaaggtg cagctggtat cagagaccat gctgctcatg gacttggtct ggaagttggt
      841 ggtcaccagg cagattcgct cctccctttg tggctccctg aacttcagtt tctggggaaa
      901 gggggggaag tccttgggca tcctgatgat aatcatgtcc ctgccgtcga tcaggtgctg
      961 ttgcagggtg gttgtgttct tcactttgaa cacgccgtgc aggctttgca ccagcagtgt
     1021 gccgttattc ctcctgaaca gatgcttgtt ggtgatgatg aaggggccga agccgatgcc
     1081 gtacagacta gtggtgtgac cgtcgctctc gttggtcagg tgacagatgg tgctggagat
     1141 cgggttgtag tccctcgggc ccttgaaaag gctctcgcca gggccgggat tctcctccac
     1201 gtcaccgcat gttagaagac ttcctctgcc ctctccgctt ccggagtgat aaaaatagag
     1261 ttcttttgtg agcatggaaa caatacatgg aatctgtttc tttgcatgaa aagtagcgtc
     1321 aaaggaaaag gactcaaatt ctgttgccac ctttcggtta acccgggtaa gaatgtgcat
     1381 aaattcaagc ttgtcggcat actgtttcag catggcacaa agcgactgga tgaaccagga
     1441 gccatccttt gaatttcgcc aagaataata accaggtgct gtggagtatg catacaagaa
     1501 gtcggcctcc actggtattt tatgacacgc catgtcatca tcaacaccac tctggaagta
     1561 gagattttct gtctcaatgc cacagtccag ttctgtacca cggcaggcct gaataatgaa
     1621 aagtttgggt tttccagtta gacttctaca acgatcccct ctgaaaaagt ttgttatttt
     1681 tttcaggtca acaggtccat ttgttccaaa aattattcct tcttcaccat ggctcagaag
     1741 cacacaaaca aaactgctcc ttttgctgtg atcttcttta gaaacatcac gcatcaattc
     1801 cacaatttct tcacgtgtaa gatcattttt attcctgact tcatatttca agtttctgaa
     1861 tgtttccctg aggtttgctg catcgacatc tgtaccagac cgagatgtca ttccagtgct
     1921 tttatgaaaa ttcttattat taattattat acataaaccc atctcaggat aatccatttt
     1981 ataactgttg tccagggata ttccagagtc cattgattcg cttccatgta tgatctttgg
     2041 ttccaaattt ttaatggatt ttgaatccac tgagttttca gtgttctcca tggtggcggc
     2101 tagcataact tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc
     2161 actgttattc tttagaatgg tgcaaagaat aacttcgtat aatgtatgct atacgaagtt
     2221 atgaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     2281 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     2341 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     2401 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     2461 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     2521 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     2581 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     2641 aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt
     2701 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     2761 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     2821 gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt ggcatccctg
     2881 tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct
     2941 tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat
     3001 ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg
     3061 cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc
     3121 cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc
     3181 atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt
     3241 ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt
     3301 gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac
     3361 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     3421 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     3481 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     3541 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     3601 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3661 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3721 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3781 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     3841 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3901 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     3961 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4021 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     4081 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4141 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4201 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     4261 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4321 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4381 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4441 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4501 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4561 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4621 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4681 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4741 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4801 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4861 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4921 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4981 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5041 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5101 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5161 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5221 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5281 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5341 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5401 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5461 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5521 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5581 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5641 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5701 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5761 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5821 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5881 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5941 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6001 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6061 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6121 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
     6181 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
     6241 aggggttcct gcggccgcac gcgtttact
//
