LOCUS       sequence-487562-        6906 bp    DNA     circular SYN 01-OCT-2025
DEFINITION  For generating adeno-associated virus to express GFE3 under the EF1a
            promoter in a 'Cre-on' dependent manner..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6906)
  AUTHORS   Gross GG, Straub C, Perez-Sanchez J, Dempsey WP, Junge JA, Roberts 
            RW, Trinh LA, Fraser SE, De Koninck Y, De Koninck P, Sabatini BL, 
            Arnold DB
  TITLE     An E3-ligase-based method for ablating inhibitory synapses.
  JOURNAL   Nat Methods. 2016 Jun 6. doi: 10.1038/nmeth.3894.
  PUBMED    27271196
REFERENCE   2  (bases 1 to 6906)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2016 Jun 6. doi: 10.1038/nmeth.3894."
COMMENT     Sequence Label: pAAV-Ef1a-DIO_EGFP-GFE3
FEATURES             Location/Qualifiers
     source          1..6906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1440..1473)
                     /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(1524..1557)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1757..1837)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(join(2165..2872,2873..2875,2876..2878,
                     2879..2881))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELY"
     CDS             complement(join(2165..2872,2873..2875,2876..2878,
                     2879..2881))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELY"
     primer_bind     complement(2204..2225)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2553..2572
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2812..2833
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2891..2924
                     /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    2975..3008
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3033..3621
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3086..3106)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3504..3515)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3653..4129
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3852..3871)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4169..4309
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4169..4298
                     /label=AAV2 ITR
     rep_origin      4384..4839
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4471..4490)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4681..4702
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4856..4875)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4975..4997
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5035..5053)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5121..5225
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5226..5294,5295..6086)
                     /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(5444..5463)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6257..6845
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6746..6765
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgccttact tgtacattaa gacataaaaa ttttttgctt gaacgtaatg actgtgcagc
     1621 acatgggaca tttgtcaact gcttccgcac actgtttaca agtgaccaga tgtccacaag
     1681 gaacaaaaac tatagcaata tttctatcca tacagatttt gcaaagcttc tcctcttgta
     1741 ggcgccttag gctagcagcg taatctggaa catcgtatgg gtaagcgtaa tctggaacat
     1801 cgtatgggta agcgtaatct ggaacatcgt atgggtaaga tcttgaaccg gtgcggtagt
     1861 tgatggagat cgggatcggg tcctggtagt tccagtggtc gatcgtgacg gcgtacaccg
     1921 tgatggtata gtcgacacca ggtttcaggc tgctgatggt agcagtggac ttgctgccag
     1981 gcacggtgaa ttcctggaca gggctattgc caccagtttc accgtaggtg atgcggtagt
     2041 agcgaaccat gaccttgtac ttgccccagc tgatctggat gctggttggt gatgcttcct
     2101 tgacttcgag caattgcgaa cctcctcctg atccacctcc agaacctcca ccagaacctc
     2161 caccgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     2221 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg ctcaggtagt
     2281 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     2341 cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt
     2401 tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt
     2461 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     2521 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     2581 agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     2641 tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg
     2701 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     2761 aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     2821 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccccca
     2881 tatgcctagc ataacttcgt ataaagtatc ctatacgaag ttatttgcct taacccagaa
     2941 attatcactg ttattcttta gaatggtgca aagaataact tcgtataatg tatgctatac
     3001 gaagttatga attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg
     3061 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     3121 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     3181 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     3241 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     3301 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     3361 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     3421 cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg attctgcgcg
     3481 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     3541 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     3601 ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatct acgggtggca
     3661 tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac
     3721 cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa
     3781 tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag
     3841 ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc
     3901 aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt
     3961 ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac
     4021 catattggcc aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc
     4081 caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgattttgta
     4141 ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc
     4201 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4261 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     4321 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     4381 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4441 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4501 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4561 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     4621 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4681 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     4741 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     4801 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     4861 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     4921 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4981 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     5041 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     5101 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5161 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5221 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5281 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5341 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5401 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5461 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5521 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5581 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5641 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5701 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5761 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5821 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5881 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5941 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6001 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6061 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6121 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6181 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6241 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6301 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6361 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6421 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6481 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6541 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6601 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6661 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6721 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6781 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6841 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6901 acatgt
//
