LOCUS       sequence-75491-e        7558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gi-coupled hM4D-IRES-mCitrine under the control of GFAP promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7558)
  AUTHORS   Bryan Roth
  TITLE     Roth lab DREADDs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7558)
  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            "Unpublished"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-GFAP-HA-hM4D(Gi)-IRES-mCitrine
FEATURES             Location/Qualifiers
     source          1..7558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     CDS             876..902
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    2358..2931
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2525..2542)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2752..2771
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2932..2941
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2938..2940,2941..2943,2944..3654)
                     /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(2983..3004)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3244..3263)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3591..3612
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3685..4273
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3738..3758)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4156..4167)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4305..4781
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4504..4523)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4821..4961
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4821..4950
                     /label=AAV2 ITR
     rep_origin      5036..5491
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5123..5142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5333..5354
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5508..5527)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5627..5649
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5687..5705)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5773..5877
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5878..5946,5947..6738)
                     /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"
     rep_origin      6909..7497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7398..7417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgtgtc tagagatcta acatatcctg
      181 gtgtggagta ggggacgctg ctctgacaga ggctcggggg cctgagctgg ctctgtgagc
      241 tggggaggag gcagacagcc aggccttgtc tgcaagcaga cctggcagca ttgggctggc
      301 cgccccccag ggcctcctct tcatgcccag tgaatgactc accttggcac agacacaatg
      361 ttcggggtgg gcacagtgcc tgcttcccgc cgcaccccag cccccctcaa atgccttccg
      421 agaagcccat tgagcagggg gcttgcattg caccccagcc tgacagcctg gcatcttggg
      481 ataaaagcag cacagccccc taggggctgc ccttgctgtg tggcgccacc ggcggtggag
      541 aacaaggctc tattcagcct gtgcccagga aaggggatca ggggatgccc aggcatggac
      601 agtgggtggc agggggggag aggagggctg tctgcttccc agaagtccaa ggacacaaat
      661 gggtgagggg agagctctcc ccatagctgg gctgcggccc aaccccaccc cctcaggcta
      721 tgccaggggg tgttgccagg ggcacccggg catcgccagt ctagcccact ccttcataaa
      781 gccctcgcat cccaggagcg agcagagcca gagcaggttg gagaggagac gcatcacctc
      841 cgctgctcgc ggggtctaga gtcgacgcca ccatgtaccc atacgatgtt ccagattacg
      901 ctatggccaa cttcacacct gtcaatggca gctcgggcaa tcagtccgtg cgcctggtca
      961 cgtcatcatc ccacaatcgc tatgagacgg tggaaatggt cttcattgcc acagtgacag
     1021 gctccctgag cctggtgact gtcgtgggca acatcctggt gatgctgtcc atcaaggtca
     1081 acaggcagct gcagacagtc aacaactact tcctcttcag cctggcgtgt gctgatctca
     1141 tcataggcgc cttctccatg aacctctaca ccgtgtacat catcaagggc tactggcccc
     1201 tgggcgccgt ggtctgcgac ctgtggctgg ccctggactg cgtggtgagc aacgcctccg
     1261 tcatgaacct tctcatcatc agctttgacc gctacttctg cgtcaccaag cctctcacct
     1321 accctgcccg gcgcaccacc aagatggcag gcctcatgat tgctgctgcc tgggtactgt
     1381 ccttcgtgct ctgggcgcct gccatcttgt tctggcagtt tgtggtgggt aagcggacgg
     1441 tgcccgacaa ccagtgcttc atccagttcc tgtccaaccc agcagtgacc tttggcacag
     1501 ccattgctgg cttctacctg cctgtggtca tcatgacggt gctgtacatc cacatctccc
     1561 tggccagtcg cagccgagtc cacaagcacc ggcccgaggg cccgaaggag aagaaagcca
     1621 agacgctggc cttcctcaag agcccactaa tgaagcagag cgtcaagaag cccccgcccg
     1681 gggaggccgc ccgggaggag ctgcgcaatg gcaagctgga ggaggccccc ccgccagcgc
     1741 tgccaccgcc accgcgcccc gtggctgata aggacacttc caatgagtcc agctcaggca
     1801 gtgccaccca gaacaccaag gaacgcccag ccacagagct gtccaccaca gaggccacca
     1861 cgcccgccat gcccgcccct cccctgcagc cgcgggccct caacccagcc tccagatggt
     1921 ccaagatcca gattgtgacg aagcagacag gcaatgagtg tgtgacagcc attgagattg
     1981 tgcctgccac gccggctggc atgcgccctg cggccaacgt ggcccgcaag ttcgccagca
     2041 tcgctcgcaa ccaggtgcgc aagaagcggc agatggcggc ccgggagcgc aaagtgacac
     2101 gaacgatctt tgccattctg cttgccttca tcctcacctg gacgccctac aacgtcatgg
     2161 tcctggtgaa caccttctgc cagagctgca tccctgacac ggtgtggtcc attggctact
     2221 ggctctgcta cgtcaacagc accatcaacc ctgcctgcta tgctctgtgc aacgccacct
     2281 ttaaaaagac cttccggcac ctgctgctgt gccagtatcg gaacatcggc actgccaggt
     2341 aggcggccgc gatatcgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc
     2401 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     2461 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     2521 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
     2581 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     2641 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     2701 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     2761 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     2821 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
     2881 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata agccaccatg
     2941 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     3001 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     3061 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     3121 gtgaccacct tcggctacgg cctgatgtgc ttcgcccgct accccgacca catgaagcag
     3181 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     3241 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     3301 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     3361 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     3421 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     3481 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     3541 ctgagctacc agtccaaact gagcaaagac cccaacgaga agcgcgatca catggtcctg
     3601 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtgatag
     3661 gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     3721 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     3781 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     3841 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     3901 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     3961 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     4021 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     4081 tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc
     4141 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     4201 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     4261 gccgcctccc cgcatcgata ccgagcgctg ctcgagagat ctacgggtgg catccctgtg
     4321 acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg
     4381 tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg
     4441 ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg
     4501 gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg
     4561 cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat
     4621 gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg
     4681 ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc
     4741 tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca
     4801 cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4861 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4921 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     4981 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     5041 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     5101 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     5161 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     5221 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     5281 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5341 ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg
     5401 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5461 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     5521 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5581 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5641 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5701 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5761 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5821 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5881 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5941 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     6001 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     6061 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     6121 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     6181 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     6241 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     6301 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6361 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6421 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6481 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6541 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6601 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6661 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6721 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6781 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     7021 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     7081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
