LOCUS       sequence-369058-        7060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Gad65, 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 7060)
  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 7060)
  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-Gad65-WPRE-HGHpA
FEATURES             Location/Qualifiers
     source          1..7060
                     /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)."
     protein_bind    5939..5972
                     /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    6023..6056
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6081..6669
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6134..6154)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6552..6563)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6703..6814
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   join(6932..7060,1..12)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(6932..7060,1)
                     /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 cgccttacaa atcttgtccg aggcgttcga
     4201 tttcttcaat aaggaagtca atgtcttggt gagttgctgc agggtttgag atgaccatgc
     4261 ggaagaagtt gaccttgtcc cctaagggtt ggtagctgac cattgtggtc ccatactcca
     4321 tcattctggc tttaatcact ggcgccacct ttgagaggcg gctcattctc tcttcattgt
     4381 cttccagagt gcgcaaacta ggaggtacaa accagaagca gacatttgtg tgttgaggct
     4441 tcccatcaaa caccatttca tagccttctc ggttttttat gatagtgtat aaatactctg
     4501 ctagctccaa acacttatca atgtgtgcct caaacccagt agtccccttt gctctccaca
     4561 tgagccataa tttaaagaca tccacatggc gtccacactg caaggccttg tctcctgtgt
     4621 cataggacag gtcatagtgc ttatcttgct gaaagaggta ggaagcatgc atctggttgc
     4681 agctctgcat cagtccctcc tctctaacca ggagagctga acactgcaag gggacaccca
     4741 tcatcttgtg aggattccat gtcacagagt tggccctctc tactccactc agcttccact
     4801 tgtgtttccg ggacatcagt aaccctccac cccaagcagc atccacgtgc atccagatct
     4861 tatacttttt gcagatgtca gctacagcca agagaggatc aaaagcccca tacacggtgg
     4921 ttccagctgt ggcactcacc aggaaaggaa caaatccttt ctgtttgact tcaaggattc
     4981 ttctttcaag atcagatgga atcattttcc ctctctcgtc acatttaatc agaatcacac
     5041 tgtctgttcc aatccctaag gctgcagctc ccttcttgag agaaaagtga ctatgctctg
     5101 atgtgaacgc gatgagcctg ggcactgctg ccatcccctt ctccttgact tctggaaaca
     5161 tcttatagcg ggcaatgagc atggcataca tgttggagat ggcgccacca ggagaaaaga
     5221 ttccatcgcc agagcccccg ggccagccaa tgatttctct cattttcttt agtgtaacat
     5281 attctagcag cacaaataca ggggcgatct cataggtgaa catgttagtg tttgctgttg
     5341 atgtcaacca gtctgctgct aatccaacca tatccaatcc agtagacagc tgattaaaat
     5401 atcggggatg ccctgtttta attgcatatt ttagagttgt ttggcaatgc gtcaaaattt
     5461 cctccagatt ttgcggttgg tctgccaatt cccaattata ctcttgaaga agctcattgg
     5521 ggtaatggaa atcgatcact ttagttgatc tatcgaaact tttcaccacg tactgaagca
     5581 aaatgttcat aacatcttgt agaaatgcca gagtgggcct ttctccatca cacgctggca
     5641 gcaggtctgt tgcgtgcaga aacgcgtagt tgacatcccc tttggggcag ttgcagggtt
     5701 tttggtcgca ggtgcaggcg accttcccgg tggcggcccg cgaggtcacg ctcccgccgc
     5761 cctccgctgg cttcccggag tctccataga gcagagcgca cagcttgttt ccgatgccgc
     5821 ccgtgaactt ttgggccacc tggcaccacg ctctcgctgt gcccgggttc tcaggatccg
     5881 cagagccatc ttcagatccg aaggaccaaa agccggagcc aggagacgcc atgctagcat
     5941 aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat tatcactgtt
     6001 attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga agttatgaat
     6061 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     6121 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     6181 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     6241 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     6301 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     6361 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     6421 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     6481 cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct
     6541 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     6601 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     6661 cctccccgca tcgataccga gcgctgctcg agagatcttc gactgtgcct tctagttgcc
     6721 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     6781 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     6841 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     6901 atgagatctc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     6961 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     7021 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
//
