LOCUS       sequence-348534-        7476 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses DREADD hM4D (Gi) only in Flp expressing cells and not in 
            Cre expressing cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7476)
  AUTHORS   Knoedler JR, Inoue S, Bayless DW, Yang T, Tantry A, Davis CH, Leung 
            NY, Parthasarathy S, Wang G, Alvarado M, Rizvi AH, Fenno LE, 
            Ramakrishnan C, Deisseroth K, Shah NM
  TITLE     A functional cellular framework for sex and estrous cycle-dependent 
            gene expression and behavior.
  JOURNAL   Cell. 2022 Feb 17;185(4):654-671.e22. doi: 
            10.1016/j.cell.2021.12.031. Epub 2022 Jan 21.
  PUBMED    35065713
REFERENCE   2  (bases 1 to 7476)
  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: 
            "10.1016/j.cell.2021.12.031"; journalName: "Cell"; date: 
            "2022-02-17- 17"; volume: "185"; issue: "4"; pages: "654-671.e22"
COMMENT     Sequence Label: pAAV-nEF-Coff/Fon DREADD Gi-mCherry
FEATURES             Location/Qualifiers
     source          1..7476
                     /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        177..388
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             401..669
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    697..730
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     primer_bind     complement(1104..1123)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1269..1302)
                     /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(1353..1386)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     primer_bind     1585..1603
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    2723..2756
                     /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    2807..2840
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     regulatory      3445..3454
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3461..3494)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     misc_feature    3603..4191
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3656..3676)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4074..4085)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4223..4699
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4422..4441)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4739..4879
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4739..4868
                     /label=AAV2 ITR
     rep_origin      4954..5409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5041..5060)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5251..5272
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5426..5445)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5545..5567
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5605..5623)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5691..5795
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5796..5864,5865..6656)
                     /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(6014..6033)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6827..7415
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7316..7335
                     /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 gcgtaaggat ctgcgatcgc tccggtgccc gtcagtgggc
      181 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg
      241 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      301 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      361 tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc
      421 acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc
      481 tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc
      541 gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac
      601 gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg
      661 ttacagatcc aagctgtgac cggcgcctac ggatccgaag ttcctattct tcaaatagta
      721 taggaacttc gcagagtagt agctggattg tagctgctat tagcaatcta aaacctctta
      781 gaagttccta ttcttcaaaa ggtataggaa cttcggcgcg ccctattact tgtacagctc
      841 gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg tactgttcca cgatggtgta
      901 gtcctcgttg tgggaggtga tgtccaactt gatgttgacg ttgtaggcgc cgggcagctg
      961 cacgggcttc ttggccttgt aggtggtctt gacctcagcg tcgtagtggc cgccgtcctt
     1021 cagcttcagc ctctgcttga tctcgccctt cagggcgccg tcctcggggt acatccgctc
     1081 ggaggaggcc tcccagccca tggtcttctt ctgcattacg gggccgtcgg aggggaagtt
     1141 ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc tgcagggagg agtcctgtgg
     1201 gacgacatga cttaaccagt taaccatgtt ctctgaacat gagaagggct ggccactctc
     1261 cagtcgacat aacttcgtat aggatacttt atacgaagtt atgcagaatg gtagctggat
     1321 tgtagctgct attagcaata tgaaacctct taataacttc gtatagtata ccttatacga
     1381 agttatacta gtcctcctgt actcacctgg gtcacggtca ccacgccgcc gtcctcgaag
     1441 ttcatcacgc gctcccactt gaagccctcg gggaaggaca gcttcaagta gtcggggatg
     1501 tcggcggggt gcttcacgta ggccttggag ccgtacatga actgagggga caggatgtcc
     1561 caggcgaagg gcagggggcc acccttggtc accttcagct tggcggtctg ggtgccctcg
     1621 taggggcggc cctcgccctc gccctcgatc tcgaactcgt ggccgttcac ggagccctcc
     1681 atgtgcacct tgaagcgcat gaactccttg atgatggcca tgttatcctc ctcgcccttg
     1741 ctcaccatgg tagcgaccgg tggatcccgc ctggcagtgc cgatgttccg atactggcac
     1801 agcagcaggt gccggaaggt ctttttaaag gtggcgttgc acagagcata gcaggcaggg
     1861 ttgatggtgc tgttgacgta gcagagccag tagccaatgg accacaccgt gtcagggatg
     1921 cagctctggc agaaggtgtt caccaggacc atgacgttgt agggcgtcca ggtgaggatg
     1981 aaggccagca gaatggcaaa gatcgttcgt gtcactttgc gctcccgggc cgccatctgc
     2041 cgcttcttgc gcacctggtt gcgagcgatg ctggcgaact tgcgggccac gttggccgca
     2101 gggcgcatgc cagccggcgt ggcaggcaca atctcaatgg ctgtcacaca ctcattgcct
     2161 gtctgcttcg tcacaatctg gatcttggac catctggagg ctgggttgag ggcccgcggc
     2221 tgcaggggag gggcgggcat ggcgggcgtg gtggcctctg tggtggacag ctctgtggct
     2281 gggcgttcct tggtgttctg ggtggcactg cctgagctgg actcattgga agtgtcctta
     2341 tcagccacgg ggcgcggtgg cggtggcagc gctggcgggg gggcctcctc cagcttgcca
     2401 ttgcgcagct cctcccgggc ggcctccccg ggcgggggct tcttgacgct ctgcttcatt
     2461 agtgggctct tgaggaaggc cagcgtcttg gctttcttct ccttcgggcc ctcgggccgg
     2521 tgcttgtgga ctcggctgcg actggccagg gagatgtgga tgtacagcac cgtcatgatg
     2581 accacaggca ggtagaagcc agcaatggct gtgccaaagc taaacacatg agaaatagag
     2641 tcataagcac atgggtcaca tacaggagat atgtatataa cattaataca attttataaa
     2701 aaaaaggggg ggcacaggta ccataacttc gtataaagta tcctatacga agttatttgc
     2761 cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa cttcgtataa
     2821 ggtatactat acgaagttat gctagcaacc ccgacactta cgtcactgct gggttggaca
     2881 ggaactggat gaagcactgg ttgtcgggca ccgtccgctt acccaccaca aactgccaga
     2941 acaagatggc aggcgcccag agcacgaagg acagtaccca ggcagcagca atcatgaggc
     3001 ctgccatctt ggtggtgcgc cgggcagggt aggtgagagg cttggtgacg cagaagtagc
     3061 ggtcaaagct gatgatgaga aggttcatga cggaggcgtt gctcaccacg cagtccaggg
     3121 ccagccacag gtcgcagacc acggcgccca ggggccagta gcccttgatg atgtacacgg
     3181 tgtagaggtt catggagaag gcgcctatga tgagatcagc acacgccagg ctgaagagga
     3241 agtagttgtt gactgtctgc agctgcctgt tgaccttgat ggacagcatc accaggatgt
     3301 tgcccacgac agtcaccagg ctcagggagc ctgtcactgt ggcaatgaag accatttcca
     3361 ccgtctcata gcgattgtgg gatgatgacg tgaccaggcg cacggactga ttgcccgagc
     3421 tgccattgac aggtgtgaag ttggccatgg tggcgagctc gaagttccta tactatttga
     3481 agaataggaa cttcttgcct taacccagaa attatcactg ttattcttta gaatggtgca
     3541 aagagaagtt cctatacctt ttgaagaata ggaacttcga attcgatatc aagcttatcg
     3601 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3661 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     3721 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     3781 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     3841 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     3901 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3961 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     4021 tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     4081 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     4141 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     4201 gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct
     4261 ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat
     4321 cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag
     4381 caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg
     4441 gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct
     4501 cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt
     4561 tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta
     4621 atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac
     4681 tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag
     4741 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     4801 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4861 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4921 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     4981 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     5041 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5101 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5161 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5221 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5281 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     5341 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     5401 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     5461 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5521 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5581 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     5641 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5701 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5761 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5821 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5881 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5941 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6001 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6061 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6121 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6181 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6241 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6301 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6361 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6421 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6481 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6541 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6601 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6661 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6721 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6781 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6841 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6901 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6961 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     7021 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7081 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7141 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7201 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7261 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7321 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7381 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7441 ggttcctggc cttttgctgg ccttttgctc acatgt
//
