LOCUS       sequence-405580-        8135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated, cre-dependent expression of the pharmacogenetic 
            actuator hM4Di-2A-dTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8135)
  TITLE     AAV plasmid for cre-dependent expression of hM3D-2A-dTomato
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8135)
  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            "Unpublished"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-hM4Di-P2A-dTomato
FEATURES             Location/Qualifiers
     source          1..8135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(178..507)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      192..327
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     492..512
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(753..1341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(833..852)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1512..2303,2304..2372))
                     /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     2135..2154
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2373..2477)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2545..2563
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2601..2623)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2723..2742
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2956..2975
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3265..3283
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3265..3282
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     repeat_region   3304..3433
                     /label=AAV2 ITR
     promoter        3739..4016
                     /label=chicken beta-actin promoter
     primer_bind     4295..4317
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4379..4398
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(4449..4482)
                     /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(4533..4566)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(4584..5285)
                     /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(4667..4690)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(4865..4884)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5134..5152
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(5286..5342)
                     /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"
     regulatory      6812..6821
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6828..6861
                     /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    6912..6945
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6970..7558
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7023..7043)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7441..7452)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7585..7602)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7591..7815
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 actagagcat ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc
       61 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
      121 accaaaggtc gcccgacgcc cgggcggcct cagtgagcga gcgagcgcgc agagcttttt
      181 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga
      241 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
      301 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
      361 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
      421 cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
      481 ggactttcca caccctaact gacacacatt ccacagctgc attaatgaat cggccaacgc
      541 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
      601 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
      661 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
      721 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
      781 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
      841 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
      901 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
      961 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     1021 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     1081 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     1141 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     1201 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     1261 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     1321 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     1381 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     1441 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     1501 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     1561 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     1621 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     1681 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     1741 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     1801 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     1861 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     1921 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     1981 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     2041 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     2101 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     2161 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     2221 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     2281 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     2341 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     2401 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     2461 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     2521 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     2581 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     2641 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     2701 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattcg
     2761 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     2821 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     2881 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     2941 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     3001 ggtgatgccg gccacgatgc gtccggcgta gaggatctgg ctagcgatga ccctgctgat
     3061 tggttcgctg accatttccg ggtgcgggac ggcgttacca gaaactcaga aggttcgtcc
     3121 aaccaaaccg actctgacgg cagtttacga gagagatgat agggtctgct tcagtaagcc
     3181 agatgctaca caattaggct tgtacatatt gtcgttagaa cgcggctaca attaatacat
     3241 aaccttatgt atcatacaca tacgatttag gtgacactat agaatacacg gaattaattc
     3301 tagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
     3361 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
     3421 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
     3481 tctaggtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     3541 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     3601 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     3661 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     3721 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
     3781 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
     3841 gggcgggggg gggggggggg cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg
     3901 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
     3961 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
     4021 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
     4081 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
     4141 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc
     4201 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg
     4261 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     4321 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     4381 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcc tctagagtcg
     4441 actccggaat aacttcgtat aggatacttt atacgaagtt atgcagaatg gtagctggat
     4501 tgtagctgct attagcaata tgaaacctct taataacttc gtatagcata cattatacga
     4561 agttatggcg cgccgtcgac ttacttgtac agctcgtcca tgccgtacag gaacaggtgg
     4621 tggcggccct cggagcgctc gtactgttcc acgatggtgt agtcctcgtt gtgggaggtg
     4681 atgtccagct tggtgtccac gtagtagtag ccgggcagtt gcacgggctt cttggccatg
     4741 tagatggtct tgaactccac caggtagtgg ccgccgtcct tcagcttcag ggcctggtgg
     4801 atctcgccct tcagcacgcc gtcgcggggg tacaggcgct cggtggaggc ctcccagccc
     4861 atggtcttct tctgcattac ggggccgtcg ggggggaagt tggtgccgcg catcttcacc
     4921 ttgtagatca gcgtgccgtc ctgcagggag gagtcctggg tcacggtcac cagaccgccg
     4981 tcctcgaagt tcatcacgcg ctcccacttg aagccctcgg ggaaggacag cttcttgtaa
     5041 tcggggatgt cggcggggtg cttcacgtac gccttggagc cgtacatgaa ctggggggac
     5101 aggatgtccc aggcgaaggg cagggggccg cccttggtca ccttcagctt ggcggtctgg
     5161 gtgccctcgt aggggcggcc ctcgccctcg ccctcgatct cgaactcgtg gccgttcatg
     5221 gagccctcca tgcgcacctt gaagcgcatg aactctttga tgacctcctc gcccttgctc
     5281 accattggcc cgggattctc ttcgacatcc cctgcttgtt tcaacaggga gaagttagtg
     5341 gctccgcttc cgttgtcttc gaaggcgacc ggttcccgcc tggcagtgcc gatgttccga
     5401 tactggcaca gcagcaggtg ccggaaggtc tttttaaagg tggcgttgca cagagcatag
     5461 caggcagggt tgatggtgct gttgacgtag cagagccagt agccaatgga ccacaccgtg
     5521 tcagggatgc agctctggca gaaggtgttc accaggacca tgacgttgta gggcgtccag
     5581 gtgaggatga aggccagcag aatggcaaag atcgttcgtg tcactttgcg ctcccgggcc
     5641 gccatctgcc gcttcttgcg cacctggttg cgagcgatgc tggcgaactt gcgggccacg
     5701 ttggccgcag ggcgcatgcc agccggcgtg gcaggcacaa tctcaatggc tgtcacacac
     5761 tcattgcctg tctgcttcgt cacaatctgg atcttggacc atctggaggc tgggttgagg
     5821 gcccgcggct gcaggggagg ggcgggcatg gcgggcgtgg tggcctctgt ggtggacagc
     5881 tctgtggctg ggcgttcctt ggtgttctgg gtggcactgc ctgagctgga ctcattggaa
     5941 gtgtccttat cagccacggg gcgcggtggc ggtggcagcg ctggcggggg ggcctcctcc
     6001 agcttgccat tgcgcagctc ctcccgggcg gcctccccgg gcgggggctt cttgacgctc
     6061 tgcttcatta gtgggctctt gaggaaggcc agcgtcttgg ctttcttctc cttcgggccc
     6121 tcgggccggt gcttgtggac tcggctgcga ctggccaggg agatgtggat gtacagcacc
     6181 gtcatgatga ccacaggcag gtagaagcca gcaatggctg tgccaaaggt cactgctggg
     6241 ttggacagga actggatgaa gcactggttg tcgggcaccg tccgcttacc caccacaaac
     6301 tgccagaaca agatggcagg cgcccagagc acgaaggaca gtacccaggc agcagcaatc
     6361 atgaggcctg ccatcttggt ggtgcgccgg gcagggtagg tgagaggctt ggtgacgcag
     6421 aagtagcggt caaagctgat gatgagaagg ttcatgacgg aggcgttgct caccacgcag
     6481 tccagggcca gccacaggtc gcagaccacg gcgcccaggg gccagtagcc cttgatgatg
     6541 tacacggtgt agaggttcat ggagaaggcg cctatgatga gatcagcaca cgccaggctg
     6601 aagaggaagt agttgttgac tgtctgcagc tgcctgttga ccttgatgga cagcatcacc
     6661 aggatgttgc ccacgacagt caccaggctc agggagcctg tcactgtggc aatgaagacc
     6721 atttccaccg tctcatagcg attgtgggat gatgacgtga ccaggcgcac ggactgattg
     6781 cccgagctgc cattgacagg tgtgaagttg gccatggtgg cgctagcata acttcgtata
     6841 aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta ttctttagaa
     6901 tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt cgatatcaag
     6961 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     7021 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     7081 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     7141 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     7201 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     7261 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     7321 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     7381 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     7441 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     7501 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     7561 cgataccgtc gactcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     7621 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     7681 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     7741 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     7801 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgact agagcatggc
     7861 tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag
     7921 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     7981 cgacgcccgg gcggcctcag tgagcgagcg agcgcgcaga gctttttgca aaagcctagg
     8041 cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc ggcctcggcc
     8101 tctgcataaa taaaaaaaat tagtcagcca tgggg
//
