LOCUS       sequence-182308-        7762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double floxed Gi-coupled hM4D DREADD fused with mCherry under the 
            control of EF1a promoter .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7762)
  AUTHORS   Bryan Roth
  TITLE     Roth lab DREADDs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7762)
  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            "Unpublished"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-EF1a-DIO-hM4D(Gi)-mCherry
FEATURES             Location/Qualifiers
     source          1..7762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      599..608
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             605..1315
                     /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(750..768)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1018..1037
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    1324..1357
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1408..1441
                     /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)."
     promoter        complement(1472..2650)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1481..2419
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(1504..1524)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   2751..2880
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2942..3530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3022..3041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3701..4492,4493..4561))
                     /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     4324..4343
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4562..4666)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4734..4752
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4790..4812)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4912..4931
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4948..5403)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5085..5106)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5297..5316
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5478..5618
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5489..5618
                     /label=AAV2 ITR
     polyA_signal    5658..6134
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     5916..5935
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    6166..6754
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6272..6283
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     6681..6701
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(6779..6812)
                     /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(6863..6896)
                     /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)."
     regulatory      6903..6912
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ccccgtggct gataaggaca cttccaatga gtccagctca ggcagtgcca cccagaacac
       61 caaggaacgc ccagccacag agctgtccac cacagaggcc accacgcccg ccatgcccgc
      121 ccctcccctg cagccgcggg ccctcaaccc agcctccaga tggtccaaga tccagattgt
      181 gacgaagcag acaggcaatg agtgtgtgac agccattgag attgtgcctg ccacgccggc
      241 tggcatgcgc cctgcggcca acgtggcccg caagttcgcc agcatcgctc gcaaccaggt
      301 gcgcaagaag cggcagatgg cggcccggga gcgcaaagtg acacgaacga tctttgccat
      361 tctgctggcc ttcatcctca cctggacgcc ctacaacgtc atggtcctgg tgaacacctt
      421 ctgccagagc tgcatccctg acacggtgtg gtccattggc tactggctct gctacgtcaa
      481 cagcaccatc aaccctgcct gctatgctct gtgcaacgcc acctttaaaa agaccttccg
      541 gcacctgctg ctgtgccagt atcggaacat cggcactgcc aggcgggatc caccggtcgc
      601 caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
      661 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
      721 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
      781 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
      841 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
      901 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
      961 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     1021 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     1081 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     1141 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     1201 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     1261 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaaggcgc
     1321 gccataactt cgtataatgt atgctatacg aagttattaa gaggtttcat attgctaata
     1381 gcagctacaa tccagctacc attctgcata acttcgtata aagtatccta tacgaagtta
     1441 ttccggagtc gactctagag gatccggtac ctcacgacac ctgaaatgga agaaaaaaac
     1501 tttgaaccac tgtctgaggc ttgagaatga accaagatcc aaactcaaaa agggcaaatt
     1561 ccaaggagaa ttacatcaag tgccaagctg gcctaacttc agtctccacc cactcagtgt
     1621 ggggaaactc catcgcataa aacccctccc cccaacctaa agacgacgta ctccaaaagc
     1681 tcgagaacta atcgaggtgc ctggacggcg cccggtactc cgtggagtca catgaagcga
     1741 cggctgagga cggaaaggcc cttttccttt gtgtgggtga ctcacccgcc cgctctcccg
     1801 agcgccgcgt cctccatttt gagctccctg cagcagggcc gggaagcggc catctttccg
     1861 ctcacgcaac tggtgccgac cgggccagcc ttgccgccca gggcggggcg atacacggcg
     1921 gcgcgaggcc aggcaccaga gcaggccggc cagcttgaga ctacccccgt ccgattctcg
     1981 gtggccgcgc tcgcaggccc cgcctcgccg aacatgtgcg ctgggacgca cgggccccgt
     2041 cgccgcccgc ggccccaaaa accgaaatac cagtgtgcag atcttggccc gcatttacaa
     2101 gactatcttg ccagaaaaaa agcgtcgcag caggtcatca aaaattttaa atggctagag
     2161 acttatcgaa agcagcgaga caggcgcgaa ggtgccacca gattcgcacg cggcggcccc
     2221 agcgcccagg ccaggcctca actcaagcac gaggcgaagg ggctccttaa gcgcaaggcc
     2281 tcgaactctc ccacccactt ccaacccgaa gctcgggatc aagaatcacg tactgcagcc
     2341 aggtggaagt aattcaaggc acgcaagggc cataacccgt aaagaggcca ggcccgcggg
     2401 aaccacacac ggcacttacc tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca
     2461 cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg cggagccagt
     2521 acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg gttcaattgc
     2581 cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc cactgacggg
     2641 caccggagcc aattcccact cctttcaaga cctagaaggt ccattagctg caaagattcc
     2701 tctctgttta aaactttatc catctttgca aagcttacgc gtgcggccgc aggaacccct
     2761 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     2821 aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     2881 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2941 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3001 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3061 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3121 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3181 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3241 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3301 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3361 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3421 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3481 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3541 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3601 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3661 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3721 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3781 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3841 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3901 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3961 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4021 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4081 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4141 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4201 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4261 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4321 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4381 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4441 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4501 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4561 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4621 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4681 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     4741 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4801 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     4861 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     4921 agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg
     4981 cgttaaattt ttgttaaatc agctcatttt ttaaccaata gaccgaaatc ggcaaaatcc
     5041 cttataaatc aaaagaatag cccgagatag agttgagtgt tgttccagtt tggaacaaga
     5101 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     5161 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag
     5221 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     5281 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctaaggcg ctggcaagtg
     5341 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     5401 cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa
     5461 ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg
     5521 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
     5581 cagagaggga gtggccaact ccatcactag gggttcctgc ggccgctcgg tccgcacgtg
     5641 gttacctaca aaatcagaag gacagggaag ggagcagtgg ttcacgcctg taatcccagc
     5701 aatttgggag gccaaggtgg gtagatcacc tgagattagg agttggagac cagcctggcc
     5761 aatatggtga aaccccgtct ctaccaaaaa aacaaaaatt agctgagcct ggtcatgcat
     5821 gcctggaatc ccaacaactc gggaggctga ggcaggagaa tcgcttgaac ccaggaggcg
     5881 gagattgcag tgagccaaga ttgtgccact gcactccagc ttggttccca atagaccccg
     5941 caggccctac aggttgtctt cccaacttgc cccttgctcc ataccacccc cctccacccc
     6001 ataatattat agaaggacac ctagtcagac aaaatgatgc aacttaattt tattaggaca
     6061 aggctggtgg gcactggagt ggcaacttcc agggccagga gaggcactgg ggaggggtca
     6121 cagggatgcc acccgtagat ctctcgagca gcgctcggta tcgatgcggg gaggcggccc
     6181 aaagggagat ccgactcgtc tgagggcgaa ggcgaagacg cggaagaggc cgcagagccg
     6241 gcagcaggcc gcgggaagga aggtccgctg gattgagggc cgaagggacg tagcagaagg
     6301 acgtcccgcg cagaatccag gtggcaacat aggcgagcag ccaaggaaag gacgatgatt
     6361 tccccgacaa caccacggaa ttgtcagtgc ccaacagccg agcccctgtc cagcagcggg
     6421 caaggcaggc ggcgatgagt tccgccgtgg caatagggag ggggaaagcg aaagtcccgg
     6481 aaaggagctg acaggtggtg gcaatgcccc aaccagtggg ggttgcgtca gcaaacacag
     6541 tgcacaccac gccacgttgc ctgacaacgg gccacaactc ctcataaaga gacagcaacc
     6601 aggatttata caaggaggag aaaatgaaag ccatacggga agcaatagca tgatacaaag
     6661 gcattaaagc agcgtatcca catagcgtaa aaggagcaac atagttaaga ataccagtca
     6721 atctttcaca aattttgtaa tccagaggtt gattatcgat aagcttgata tcgaattcat
     6781 aacttcgtat agcatacatt atacgaagtt attctttgca ccattctaaa gaataacagt
     6841 gataatttct gggttaaggc aaataacttc gtataggata ctttatacga agttatgcta
     6901 gcgccaccat ggccaacttc acacctgtca atggcagctc gggcaatcag tccgtgcgcc
     6961 tggtcacgtc atcatcccac aatcgctatg agacggtgga aatggtcttc attgccacag
     7021 tgacaggctc cctgagcctg gtgactgtcg tgggcaacat cctggtgatg ctgtccatca
     7081 aggtcaacag gcagctgcag acagtcaaca actacttcct cttcagcctg gcgtgtgctg
     7141 atctcatcat aggcgccttc tccatgaacc tctacaccgt gtacatcatc aagggctact
     7201 ggcccctggg cgccgtggtc tgcgacctgt ggctggccct ggactgcgtg gtgagcaacg
     7261 cctccgtcat gaaccttctc atcatcagct ttgaccgcta cttctgcgtc accaagcctc
     7321 tcacctaccc tgcccggcgc accaccaaga tggcaggcct catgattgct gctgcctggg
     7381 tactgtcctt cgtgctctgg gcgcctgcca tcttgttctg gcagtttgtg gtgggtaagc
     7441 ggacggtgcc cgacaaccag tgcttcatcc agttcctgtc caacccagca gtgacctttg
     7501 gcacagccat tgctggcttc tacctgcctg tggtcatcat gacggtgctg tacatccaca
     7561 tctccctggc cagtcgcagc cgagtccaca agcaccggcc cgagggcccg aaggagaaga
     7621 aagccaagac gctggccttc ctcaagagcc cactaatgaa gcagagcgtc aagaagcccc
     7681 cgcccgggga ggccgcccgg gaggagctgc gcaatggcaa gctggaggag gcccccccgc
     7741 cagcgctgcc accgccaccg cg
//
