LOCUS       sequence-369063-        7199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the GABA membrane transporter Gat1 R69K mutant fused to 
            HA, driven by the Ef1a promoter, in a Cre-dependent fashion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7199)
  AUTHORS   Melani R, Tritsch NX
  TITLE     Inhibitory co-transmission from midbrain dopamine neurons relies on 
            presynaptic GABA uptake.
  JOURNAL   Cell Rep. 2022 Apr 19;39(3):110716. doi: 
            10.1016/j.celrep.2022.110716.
  PUBMED    35443174
REFERENCE   2  (bases 1 to 7199)
  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: "Cell Rep.";
            date: "2022-04-19"; volume: "39(3):110716. doi"; pages: " 
            10.1016/j.celrep.2022.110716"
COMMENT     Sequence Label: pAAV-EF1a-double floxed-Gat1(R69K)-HA-WPRE-HGHpA
FEATURES             Location/Qualifiers
     source          1..7199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      87..542
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(174..193)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     384..405
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(559..578)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     678..700
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(738..756)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        824..928
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(929..997,998..1789)
                     /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(1147..1166)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1960..2548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2449..2468
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2610..2739
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2840..4018
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3071..4009
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3966..3986
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(4049..4082)
                     /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(4133..4166)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(4178..4267)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     regulatory      6064..6073
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6078..6111
                     /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    6162..6195
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6220..6808
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6273..6293)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6691..6702)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6842..6953
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   join(7071..7199,1..12)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(7071..7199,1)
                     /label=AAV2 ITR
ORIGIN
        1 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
       61 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
      121 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
      181 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      241 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
      301 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
      361 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
      421 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
      481 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
      541 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
      601 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
      661 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
      721 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
      781 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      841 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      901 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      961 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     1021 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1081 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1141 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1201 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1261 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1321 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1381 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1441 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1501 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1561 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1621 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1681 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1741 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1801 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1861 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1921 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1981 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2041 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2101 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2161 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2221 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2281 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2341 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2401 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2461 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2521 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2581 ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac
     2641 tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
     2701 gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg cgtaagcttt
     2761 gcaaagatgg ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt
     2821 cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
     2881 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
     2941 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     3001 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     3061 cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg
     3121 cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct
     3181 tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg
     3241 tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct
     3301 tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc
     3361 tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg
     3421 tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc
     3481 ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg
     3541 ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag
     3601 gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc
     3661 agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca
     3721 aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc
     3781 gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg
     3841 ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc
     3901 agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt
     3961 cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgagg
     4021 taccggatcc tctagagtcg actccggaat aacttcgtat aggatacttt atacgaagtt
     4081 atgcagaatg gtagctggat tgtagctgct attagcaata tgaaacctct taataacttc
     4141 gtatagcata cattatacga agttatggcg cgccctaagc gtaatctgga acgtcatatg
     4201 gataggatcc tgcatagtcc gggacgtcat agggatagcc agcgtaatct ggaacatcgt
     4261 atgggtagcc gatgtaggcc tccttgctgg ctgagctgcc agcctgcggc tgctcagggc
     4321 cattctcagg gcgcacaata tcttcactgg gctgaatcat gacctggaga cgctgcttca
     4381 gggagccctt cagggtgagg aacatgtaag ccatgtaccc ggggatgagc accatggagg
     4441 acagagccat gagccagccc acgccctggc cccacttggg gaaaacatag cttcccatgg
     4501 tgagtggtgt catctgcaca gcactgaaga gaaacacgcc cgccacaatg atgggtgtga
     4561 aaaaggacca gcacagcttc caccagatgc agggcctgga gccaaccatc tcctggatgt
     4621 tgtcatagaa ccggttgaca ccataaaacc aggaaatgga gacacactcg aagaaaacca
     4681 ggaacagcaa gctcatgccg ctggcagagt aataatcaaa cagtttgaag acataaatgc
     4741 caccctgggt gatgttagac aggccaatca ggtaggacac gatgcacacg gcagcaatga
     4801 agagttcacg gcgattgcgg agaagtctgg ggtactcgtc caccagggca gtgatgaagc
     4861 cctccacggt acagaactgg ctgtcaatgc ccagcatcag cagcatggag aagaagagga
     4921 tagcccagag gggagagatg ggtagctgtg tcacagcctc agggtacgcc aagaatgcca
     4981 gccccgggcc tgaggctgcc acatcagcta tggacctctt ggtgacatga gccatgaagc
     5041 ccacgatgga gaagatgacg aatccggcaa acatgctggt gcaggagttg atgcagcaaa
     5101 cgatgatgga gtccctgtac acattgttgt ggaaagagtt gtagcttccc agagcaatca
     5161 gggaccccag gcccagcccg taggagaaga agatctgggt ggcggcgtca agccacacct
     5221 cagaatcaga cagctttcgg aagttgggtg tgatgtagaa gaggatcccc tccttggccc
     5281 cgggaagcgt cactccacgg aagaacagga tgataagcat gatgtagggg tacgtggctg
     5341 agaagtagac cacctttcca gtccaaccaa cacccttcca gatgcagaaa tacacgagca
     5401 cccaggcaat ggccagtgtg atggccagag gccagcggat ctgtcctggc ttgtccagtc
     5461 catctgtcat ctggtgcatg ttgcgctccc agaactccac cacggcgctg gtcatgttgg
     5521 tggtattgac caggctgtag ttggagaagc agcggtcagt gttccacggg ttgtcacact
     5581 gtttccatgg cagggtcgtg gtgaaggagt tgtacaggta gtagatggcc caggagatga
     5641 tgacgatgta gtagatgttc agccagaagg acagcacagc tgccgcgagg cccacaccct
     5701 tgaacatggg cgccagcttc catacgccca ggcccccaat ggaggtgtac tggcctaggg
     5761 agcactccaa aaggaagaga ggaacacccg caaagatgag cgtcaggaaa tatgggatta
     5821 ggaaggcccc gccaccgttt ttcccacaga ggtaagggaa cttccacaca ttgcccaggc
     5881 cgatggcata gcccacgcag gacatgagga agtcgaagcg tcccttccat gtgtcccggt
     5941 cagggaggtc cccggccttc ttctgcacct tgactaccag ggttttgggc ttgtcgctgg
     6001 ccacaggggc ctcgctgacc tcagtagaga tctgcccatc agccaccttg ctgttgtcag
     6061 tcgccatggt ggctagcata acttcgtata aagtatccta tacgaagtta tttgccttaa
     6121 cccagaaatt atcactgtta ttctttagaa tggtgcaaag aataacttcg tataatgtat
     6181 gctatacgaa gttatgaatt cgatatcaag cttatcgata atcaacctct ggattacaaa
     6241 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     6301 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     6361 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     6421 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     6481 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     6541 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     6601 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc cacctggatt
     6661 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     6721 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     6781 cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga gagatcttcg
     6841 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     6901 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     6961 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     7021 tgggaagaca atagcaggca tgagatctca cgtgcggacc gagcggccgc aggaacccct
     7081 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     7141 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgca
//
