LOCUS       sequence-454100-        6961 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For AAV packaging and Cre-dependent expression of a peripherally 
            restricted DREADD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6961)
  AUTHORS   Kang HJ, Krumm BE, Tassou A, Geron M, DiBerto JF, Kapolka NJ, 
            Gumpper RH, Sakamoto K, Dewran Kocak D, Olsen RHJ, Huang XP, Zhang 
            S, Huang KL, Zaidi SA, Nguyen MT, Jo MJ, Katritch V, Fay JF, 
            Scherrer G, Roth BL
  TITLE     Structure-guided design of a peripherally restricted chemogenetic 
            system.
  JOURNAL   Cell. 2024 Dec 26;187(26):7433-7449.e20. doi: 
            10.1016/j.cell.2024.11.001. Epub 2024 Dec 3.
  PUBMED    39631393
REFERENCE   2  (bases 1 to 6961)
  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.2024.11.001"; journalName: "Cell"; date: 
            "2024-12-26- 26"; volume: "187"; issue: "26"; pages: "7433-7449.e20"
COMMENT     Sequence Label: pAAV-CAG-DIO-HA-HCARD-P2A-mCitrine
FEATURES             Location/Qualifiers
     source          1..6961
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        223..526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    complement(945..978)
                     /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(1029..1062)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1074..1784,1785..1787,1788..1790))
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1116..1137)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1465..1484
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1724..1745
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1787..1796
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1797..1853)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(2934..2960)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    2974..3007
                     /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    3058..3091
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3116..3704
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3169..3189)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3587..3598)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3730..3747)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3736..3943
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3973..4113
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3973..4102
                     /label=AAV2 ITR
     rep_origin      4199..4654
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4286..4305)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4496..4517
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4721..4740)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4840..4862
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4900..4918)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4986..5090
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5091..5159,5160..5951)
                     /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(5309..5328)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6122..6710
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6611..6630
                     /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 gaattcggta cctagttatt aatagtaatc
      181 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      241 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      301 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      361 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      421 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      481 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      541 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      601 tattttttaa ttattttgtg cagcgatggg gggggggggg gggggggggg gggggggggg
      661 cggggggggg gggggggggg gggggggggg gggggggcgg agaggtgcgg cggcagccaa
      721 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      781 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
      841 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
      901 gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgctataact tcgtatagga
      961 tactttatac gaagttatgc agaatggtag ctggattgta gctgctatta gcaatatgaa
     1021 acctcttaat aacttcgtat agcatacatt atacgaagtt atggcgcgcc tcacttgtac
     1081 agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga
     1141 tcgcgcttct cgttggggtc tttgctcagt ttggactggt agctcaggta gtggttgtcg
     1201 ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc
     1261 acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc
     1321 ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg
     1381 atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg
     1441 ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg
     1501 cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg
     1561 tcggggtagc gggcgaagca catcaggccg tagccgaagg tggtcacgag ggtgggccag
     1621 ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca
     1681 tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg
     1741 accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat ggtggcaggt
     1801 ccagggttct cctccacgtc tccagcctgc ttcagcaggc tgaagttagt agcgcgggac
     1861 gtagaagcga ggtacggggg actgccaggt tcgctagggt ccgccataag agcaccaggt
     1921 attgatctcg ttgttgatgg gtctccagta agttctacac tggtggatcg gttgttatcg
     1981 ggctcaccaa gagttttctt gcgcaaacat ctgtttatgc aggtactaaa gaaatttgga
     2041 aagctcggac tgctaaaata gtatacaacg ggatcgagca ttgagttcat ataggtgaag
     2101 ctgagtgttg taaaaaaagc gaggtctaca ctactgtaaa tatcacagtt ccgaacattg
     2161 tacttgtaca gaagccagaa gattcgaatg cgtacagcga cgcttggaag gaagcatata
     2221 ataaaaacta ttgccactac cattatgaaa ttgattgccc gtttgatctt ggcgtgtcta
     2281 tccatctgtc tttgccgcag ggaccaaatg atgcgaccag agcaaaagag gatgatagcc
     2341 aggggaagaa aaaattcgag gagaaacata gcatcatgcc acctaaagtt gtaacatata
     2401 gaaaatgaac tgcacagata ggcttctccg ttcttggtca tcatgtttgt atacagcaag
     2461 tggacggtaa gcccaatcgt caatccccac agaaagcagc taataatagc agcagtgcgg
     2521 tttgatattt tattaagaaa atgatgcgga tgaactaccc taaaatacct atcgaccgct
     2581 accaccgtca agaagatgat tgaaccctgt ttgttcatgg ccagcatgaa gagcattacg
     2641 cgacacggga tcccgccaaa ccgccagtcc cagttatgta catagttatc tgtcagaaac
     2701 ggcaggcata tgatgagaag aaaatctgcg acggcgagat taaacaagaa tattcggctt
     2761 gatttccagg acttcagatg gaagcaaaat atccagagcg caagtccgtt tccaagcaat
     2821 ccaaagacaa attccagtcc cagtaccggg ggaagcactt ttgctatatt ttcgtctcta
     2881 aacacacagc agttctttcc attgataacc aagaaatggt cagacttaga catagcgtaa
     2941 tctggaacat cgtatgggta catggtggct agcataactt cgtataaagt atcctatacg
     3001 aagttatttg ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagaata
     3061 acttcgtata atgtatgcta tacgaagtta tgaattcgat atcaagctta tcgataatca
     3121 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     3181 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     3241 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     3301 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     3361 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     3421 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     3481 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta
     3541 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     3601 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     3661 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accggagctc
     3721 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     3781 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     3841 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     3901 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggagagatct cacgtgcgga
     3961 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     4021 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4081 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgccagct ggcgtaatag
     4141 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac
     4201 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4261 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4321 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4381 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4441 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4501 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4561 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4621 gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt ctccttacgc
     4681 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     4741 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4801 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4861 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     4921 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     4981 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5041 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5101 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5161 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5221 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5281 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5341 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5401 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5461 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5521 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5581 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5641 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5701 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5761 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5821 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5881 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5941 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6001 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6061 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6121 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6181 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6241 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6301 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6361 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6421 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6481 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6541 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6601 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6661 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6721 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6781 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6841 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     6901 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg ggctcacatg
     6961 t
//
