LOCUS       sequence-405528-        8180 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated, cre-dependent expression of the pharmacogenetic 
            actuator hM3Dq-2A-dTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8180)
  TITLE     AAV plasmid for cre-dependent expression of hM3D-2A-dTomato
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8180)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-DIO-CAG-hM3Dq-P2A-dTomato
FEATURES             Location/Qualifiers
     source          1..8180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     350..370
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(448..481)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(532..565)
                     /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)."
     CDS             2384..2440
                     /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             2441..3142
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(2574..2592)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2842..2861
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3036..3059
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    3160..3193
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3244..3277
                     /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)."
     primer_bind     complement(3409..3431)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     repeat_region   4293..4422
                     /label=AAV2 ITR
     promoter        complement(4443..4461)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4444..4461)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4751..4770)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(4984..5003)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5103..5125
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5249..5353
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5354..5422,5423..6214)
                     /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(5572..5591)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6385..6973
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6874..6893
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(7214..7234)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7219..7548
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7399..7534
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    7758..7982
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7971..7988
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     misc_feature    join(8015..8180,1..423)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             8121..8132
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 ggcgagcagc caaggaaagg acgatgattt ccccgacaac accacggaat tgtcagtgcc
       61 caacagccga gcccctgtcc agcagcgggc aaggcaggcg gcgatgagtt ccgccgtggc
      121 aatagggagg gggaaagcga aagtcccgga aaggagctga caggtggtgg caatgcccca
      181 accagtgggg gttgcgtcag caaacacagt gcacaccacg ccacgttgcc tgacaacggg
      241 ccacaactcc tcataaagag acagcaacca ggatttatac aaggaggaga aaatgaaagc
      301 catacgggaa gcaatagcat gatacaaagg cattaaagca gcgtatccac atagcgtaaa
      361 aggagcaaca tagttaagaa taccagtcaa tctttcacaa attttgtaat ccagaggttg
      421 attatcgata agcttgatat cgaattcata acttcgtata gcatacatta tacgaagtta
      481 ttctttgcac cattctaaag aataacagtg ataatttctg ggttaaggca aataacttcg
      541 tataggatac tttatacgaa gttatgctag cgccaccatg accttgcaca ataacagtac
      601 aacctcgcct ttgtttccaa acatcagctc ctcctggata cacagcccct ccgatgcagg
      661 gctgcccccg ggaaccgtca ctcatttcgg cagctacaat gtttctcgag cagctggcaa
      721 tttctcctct ccagacggta ccaccgatga ccctctggga ggtcataccg tctggcaagt
      781 ggtcttcatc gctttcttaa cgggcatcct ggccttggtg accatcatcg gcaacatcct
      841 ggtaattgtg tcatttaagg tcaacaagca gctgaagacg gtcaacaact acttcctctt
      901 aagcctggcc tgtgccgatc tgattatcgg ggtcatttca atgaatctgt ttacgaccta
      961 catcatcatg aatcgatggg ccttagggaa cttggcctgt gacctctggc ttgccattga
     1021 ctgcgtagcc agcaatgcct ctgttatgaa tcttctggtc atcagctttg acagatactt
     1081 ttccatcacg aggccgctca cgtaccgagc caaacgaaca acaaagagag ccggtgtgat
     1141 gatcggtctg gcttgggtca tctcctttgt cctttgggct cctgccatct tgttctggca
     1201 atactttgtt ggaaagagaa ctgtgcctcc gggagagtgc ttcattcagt tcctcagtga
     1261 gcccaccatt acttttggca cagccatcgc tggtttttat atgcctgtca ccattatgac
     1321 tattttatac tggaggatct ataaggaaac tgaaaagcgt accaaagagc ttgctggcct
     1381 gcaagcctct gggacagagg cagagacaga aaactttgtc caccccacgg gcagttctcg
     1441 aagctgcagc agttacgaac ttcaacagca aagcatgaaa cgctccaaca ggaggaagta
     1501 tggccgctgc cacttctggt tcacaaccaa gagctggaaa cccagctccg agcagatgga
     1561 ccaagaccac agcagcagtg acagttggaa caacaatgat gctgctgcct ccctggagaa
     1621 ctccgcctcc tccgacgagg aggacattgg ctccgagacg agagccatct actccatcgt
     1681 gctcaagctt ccgggtcaca gcaccatcct caactccacc aagttaccct catcggacaa
     1741 cctgcaggtg cctgaggagg agctggggat ggtggacttg gagaggaaag ccgacaagct
     1801 gcaggcccag aagagcgtgg acgatggagg cagttttcca aaaagcttct ccaagcttcc
     1861 catccagcta gagtcagccg tggacacagc taagacttct gacgtcaact cctcagtggg
     1921 taagagcacg gccactctac ctctgtcctt caaggaagcc actctggcca agaggtttgc
     1981 tctgaagacc agaagtcaga tcactaagcg gaaaaggatg tccctggtca aggagaagaa
     2041 agcggcccag accctcagtg cgatcttgct tgccttcatc atcacttgga ccccatacaa
     2101 catcatggtt ctggtgaaca ccttttgtga cagctgcata cccaaaacct tttggaatct
     2161 gggctactgg ctgtgctaca tcaacagcac cgtgaacccc gtgtgctatg ctctgtgcaa
     2221 caaaacattc agaaccactt tcaagatgct gctgctgtgc cagtgtgaca aaaaaaagag
     2281 gcgcaagcag cagtaccagc agagacagtc ggtcattttt cacaagcgcg cacccgagca
     2341 ggccttgaag gaaccggtcg ccttcgaaga caacggaagc ggagccacta acttctccct
     2401 gttgaaacaa gcaggggatg tcgaagagaa tcccgggcca atggtgagca agggcgagga
     2461 ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag ggctccatga acggccacga
     2521 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     2581 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcat
     2641 gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc cccgattaca agaagctgtc
     2701 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gtctggtgac
     2761 cgtgacccag gactcctccc tgcaggacgg cacgctgatc tacaaggtga agatgcgcgg
     2821 caccaacttc ccccccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     2881 caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccacc aggccctgaa
     2941 gctgaaggac ggcggccact acctggtgga gttcaagacc atctacatgg ccaagaagcc
     3001 cgtgcaactg cccggctact actacgtgga caccaagctg gacatcacct cccacaacga
     3061 ggactacacc atcgtggaac agtacgagcg ctccgagggc cgccaccacc tgttcctgta
     3121 cggcatggac gagctgtaca agtaagtcga cggcgcgcca taacttcgta taatgtatgc
     3181 tatacgaagt tattaagagg tttcatattg ctaatagcag ctacaatcca gctaccattc
     3241 tgcataactt cgtataaagt atcctatacg aagttattcc ggagtcgact ctagaggatc
     3301 caattctttg ccaaaatgat gagacagcac aataaccagc acgttgccca ggagctgtag
     3361 gaaaaagaag aaggcatgaa catggttagc agaggctcta gagccgccgg tcacacgcca
     3421 gaagccgaac cccgccctgc cccgtccccc ccgaaggcag ccgtcccccc gcggacagcc
     3481 ccgagccgct ccccccgcac aaagggccct cccggagccc ctcaaggctt tcacgcagcc
     3541 acagaaaaga aacaagccgt cattaaacca agcgctaatt acagcccgga ggagaagggc
     3601 cgtcccgccc gctcacctgt gggagtaacg cggtcagtca gagccggggc gggcggcgcg
     3661 aggcggcggc ggagcggggc acggggcgaa ggcagcgcgc agcgactccc gcccgccgcg
     3721 cgcttcgctt tttatagggc cgccgccgcc gccgcctcgc cataaaagga aactttcgga
     3781 gcgcgccgct ctgattggct gccgccgcmc ctctccsccy csccccsccc csccccccsc
     3841 cccccccccc ccccccyggc gcgcgccccc cccccccccc cgcccccatc gctgcacaaa
     3901 ataattaaaa aataaataaa tacaaaattg ggggtgggga ggggggggag atggggagag
     3961 tgaagcagaa cgtggggctc acctcgacca tggtaatagc gatgactaat acgtagatgt
     4021 actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
     4081 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
     4141 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
     4201 ttactatggg aacatacgtc attattgacg tcaatggtac ctagagcatg gctacgtaga
     4261 taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac
     4321 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4381 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctagaatt aattccgtgt
     4441 attctatagt gtcacctaaa tcgtatgtgt atgatacata aggttatgta ttaattgtag
     4501 ccgcgttcta acgacaatat gtacaagcct aattgtgtag catctggctt actgaagcag
     4561 accctatcat ctctctcgta aactgccgtc agagtcggtt tggttggacg aaccttctga
     4621 gtttctggta acgccgtccc gcacccggaa atggtcagcg aaccaatcag cagggtcatc
     4681 gctagccaga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     4741 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     4801 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     4861 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     4921 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaat ggtgcactct
     4981 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     5041 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5101 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     5161 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     5221 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5281 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5341 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5401 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5461 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5521 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5581 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5641 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5701 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5761 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5821 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5881 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5941 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6001 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6061 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6121 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6181 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6241 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6301 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6361 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6421 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6481 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6541 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6601 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6661 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6721 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6781 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6841 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6901 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6961 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7021 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     7081 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     7141 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     7201 ttaatgcagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     7261 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     7321 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     7381 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     7441 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     7501 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctctgcgcg
     7561 ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
     7621 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc cttgtagtta
     7681 atgattaacc cgccatgcta cttatctacg tagccatgct ctagtcgagc cccagctggt
     7741 tctttccgcc tcagaagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc
     7801 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
     7861 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
     7921 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     7981 agtcgaggct gatcagcgag tcgacggtat cgatgcgggg aggcggccca aagggagatc
     8041 cgactcgtct gagggcgaag gcgaagacgc ggaagaggcc gcagagccgg cagcaggccg
     8101 cgggaaggaa ggtccgctgg attgagggcc gaagggacgt agcagaagga cgtcccgcgc
     8161 agaatccagg tggcaacaca
//
