LOCUS       sequence-337036-        6236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Activity-dependent, cre-dependent, temporally restricted inhibitory 
            DREADD expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6236)
  AUTHORS   Rajebhosale P, Ananth MR, Kim R, Crouse RB, Jiang L, Lopez-Hernandez
            G, Zhong C, Arty C, Wang S, Jone A, Desai NS, Li Y, Picciotto MR, 
            Role LW, Talmage DA
  TITLE     Functionally refined encoding of threat memory by distinct 
            populations of basal forebrain cholinergic projection neurons.
  JOURNAL   Elife. 2024 Feb 16;13:e86581. doi: 10.7554/eLife.86581.
  PUBMED    38363713
REFERENCE   2  (bases 1 to 6236)
  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: "Elife."; 
            date: "2024-02-16"; volume: "13:e86581. doi"; pages: " 
            10.7554/eLife.86581"
COMMENT     Sequence Label: pAAV-6xTRE-FLEX-hM4Di.mCherry
FEATURES             Location/Qualifiers
     source          1..6236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   43..172
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_feature    187..775
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             293..304
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     702..722
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(794..827)
                     /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(838..871)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      890..899
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2348..2357
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2354..3064
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2499..2517)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2767..2786
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    3075..3108
                     /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    3119..3152
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3231..3249
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3267..3285
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3303..3321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3339..3357
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3374..3392
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3410..3428
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     repeat_region   3541..3681
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3541..3670
                     /label=AAV2 ITR
     rep_origin      3756..4211
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3843..3862)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4053..4074
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4228..4247)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4347..4369
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4407..4425)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4493..4597
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4598..4666,4667..5458)
                     /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(4816..4835)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5629..6217
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6118..6137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg
       61 ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
      121 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc
      181 gatcaggcgg ggaggcggcc caaagggaga tccgactcgt ctgagggcga aggcgaagac
      241 gcggaagagg ccgcagagcc ggcagcaggc cgcgggaagg aaggtccgct ggattgaggg
      301 ccgaagggac gtagcagaag gacgtcccgc gcagaatcca ggtggcaaca caggcgagca
      361 gccaaggaaa ggacgatgat ttccccgaca acaccacgga attgtcagtg cccaacagcc
      421 gagcccctgt ccagcagcgg gcaaggcagg cggcgatgag ttccgccgtg gcaataggga
      481 gggggaaagc gaaagtcccg gaaaggagct gacaggtggt ggcaatgccc caaccagtgg
      541 gggttgcgtc agcaaacaca gtgcacacca cgccacgttg cctgacaacg ggccacaact
      601 cctcataaag agacagcaac caggatttat acaaggagga gaaaatgaaa gccatacggg
      661 aagcaatagc atgatacaaa ggcattaaag cagcgtatcc acatagcgta aaaggagcaa
      721 catagttaag aataccagtc aatctttcac aaattttgta atccagaggt tgattatgga
      781 tctatgatct tcgataactt cgtataggat actttatacg aagttattgc gattcggata
      841 acttcgtata gcatacatta tacgaagtta tcaaggccgg ccggatctcg ccaccatggc
      901 caacttcaca cctgtcaatg gcagctcggg caatcagtcc gtgcgcctgg tcacgtcatc
      961 atcccacaat cgctatgaga cggtggaaat ggtcttcatt gccacagtga caggctccct
     1021 gagcctggtg actgtcgtgg gcaacatcct ggtgatgctg tccatcaagg tcaacaggca
     1081 gctgcagaca gtcaacaact acttcctctt cagcctggcg tgtgctgatc tcatcatagg
     1141 cgccttctcc atgaacctct acaccgtgta catcatcaag ggctactggc ccctgggcgc
     1201 cgtggtctgc gacctgtggc tggccctgga ctgcgtggtg agcaacgcct ccgtcatgaa
     1261 ccttctcatc atcagctttg accgctactt ctgcgtcacc aagcctctca cctaccctgc
     1321 ccggcgcacc accaagatgg caggcctcat gattgctgct gcctgggtac tgtccttcgt
     1381 gctctgggcg cctgccatct tgttctggca gtttgtggtg ggtaagcgga cggtgcccga
     1441 caaccagtgc ttcatccagt tcctgtccaa cccagcagtg acctttggca cagccattgc
     1501 tggcttctac ctgcctgtgg tcatcatgac ggtgctgtac atccacatct ccctggccag
     1561 tcgcagccga gtccacaagc accggcccga gggcccgaag gagaagaaag ccaagacgct
     1621 ggccttcctc aagagcccac taatgaagca gagcgtcaag aagcccccgc ccggggaggc
     1681 cgcccgggag gagctgcgca atggcaagct ggaggaggcc cccccgccag cgctgccacc
     1741 gccaccgcgc cccgtggctg ataaggacac ttccaatgag tccagctcag gcagtgccac
     1801 ccagaacacc aaggaacgcc cagccacaga gctgtccacc acagaggcca ccacgcccgc
     1861 catgcccgcc cctcccctgc agccgcgggc cctcaaccca gcctccagat ggtccaagat
     1921 ccagattgtg acgaagcaga caggcaatga gtgtgtgaca gccattgaga ttgtgcctgc
     1981 cacgccggct ggcatgcgcc ctgcggccaa cgtggcccgc aagttcgcca gcatcgctcg
     2041 caaccaggtg cgcaagaagc ggcagatggc ggcccgggag cgcaaagtga cacgaacgat
     2101 ctttgccatt ctgctggcct tcatcctcac ctggacgccc tacaacgtca tggtcctggt
     2161 gaacaccttc tgccagagct gcatccctga cacggtgtgg tccattggct actggctctg
     2221 ctacgtcaac agcaccatca accctgcctg ctatgctctg tgcaacgcca cctttaaaaa
     2281 gaccttccgg cacctgctgc tgtgccagta tcggaacatc ggcactgcca ggcgggatcc
     2341 accggtcgcc accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     2401 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     2461 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     2521 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     2581 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     2641 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     2701 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     2761 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     2821 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     2881 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     2941 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     3001 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     3061 gtaaggcgcg ccaaataact tcgtataaag tatcctatac gaagttattg cgattcggat
     3121 aacttcgtat aatgtatgct atacgaagtt atcgacgttc tgacggttca ctaaacgagc
     3181 tctgctttta taggcgccca ccgtacacgc ctaaagctct ggtccgtata tctctatcac
     3241 tgatagggag ttcactcctt atcagttctc tatcactgat agggagtaaa gtctgcatac
     3301 gttctctatc actgataggg agtacactct tcatagcttc tctatcactg atagggagta
     3361 ttagcttata gcttctctat cactgatagg gagtaaactg gatataattt ctctatcact
     3421 gatagggagt ttactgtaga tacgttctct atcactgata ggagtaaact aagggcgaat
     3481 tctgcagata tccatcacac tggcggccgt tactagtgga tctcgagagt tggcggccgc
     3541 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     3601 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     3661 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     3721 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     3781 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     3841 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3901 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3961 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     4021 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     4081 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     4141 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     4201 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4261 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4321 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4381 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4441 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4501 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4561 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4621 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4681 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4741 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4801 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4861 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4921 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4981 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     5041 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     5101 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     5161 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5221 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5281 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5341 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5401 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5461 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5521 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5581 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5641 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5701 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5761 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5821 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5881 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5941 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6001 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     6061 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggcct
//
