LOCUS       sequence-149707-        6910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For generating adeno-associated virus to express RandE3 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 6910)
  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 6910)
  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: "Nat 
            Methods. 2016 Jun 6. doi: 10.1038/nmeth.3894."
COMMENT     Sequence Label: pAAV-Ef1a-DIO_EGFP-RandE3
FEATURES             Location/Qualifiers
     source          1..6910
                     /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
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1450..1483)
                     /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(1534..1567)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1767..1847)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(join(2169..2876,2877..2879,2880..2882,
                     2883..2885))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELY"
     CDS             complement(join(2169..2876,2877..2879,2880..2882,
                     2883..2885))
                     /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(2208..2229)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2557..2576
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2816..2837
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    2895..2928
                     /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    2979..3012
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3037..3625
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3090..3110)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3508..3519)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3657..4133
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3856..3875)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4173..4313
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4173..4302
                     /label=AAV2 ITR
     rep_origin      4388..4843
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4475..4494)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4685..4706
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4860..4879)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4979..5001
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5039..5057)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5125..5229
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5230..5298,5299..6090)
                     /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(5448..5467)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6261..6849
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6750..6769
                     /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 cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgccttact tgtacattaa gacataaaaa ttttttgctt gaacgtaatg
     1621 actgtgcagc acatgggaca tttgtcaact gcttccgcac actgtttaca agtgaccaga
     1681 tgtccacaag gaacaaaaac tatagcaata tttctatcca tacagatttt gcaaagcttc
     1741 tcctcttgta ggcgccttag gctagcagcg taatctggaa catcgtatgg gtaagcgtaa
     1801 tctggaacat cgtatgggta agcgtaatct ggaacatcgt atgggtaaga tctggtgcgg
     1861 tagttgatgg agatcgggga ctggtcggag ctggcctggg gctcgcgcgt gacggcgtac
     1921 accgtgatgg tatagtcgac accaggtttc aggccgctga tggtagcagt ggacttgctg
     1981 ccaggcacgg tgaattcctg gacagggcta ttgccaccag tttcaccgta ggtgatgcgg
     2041 tagtagcgcg cgtgctcctg cgtcgcccgc cagctgatta ggaggctggt cggagtcgca
     2101 acgacgactt cgagcaattg cgaacctcct cctgatccac ctccagaacc tccaccagaa
     2161 cctccaccgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     2221 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg
     2281 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     2341 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg
     2401 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
     2461 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
     2521 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
     2581 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
     2641 tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg
     2701 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
     2761 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
     2821 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
     2881 cccatatgcc tagcataact tcgtataaag tatcctatac gaagttattt gccttaaccc
     2941 agaaattatc actgttattc tttagaatgg tgcaaagaat aacttcgtat aatgtatgct
     3001 atacgaagtt atgaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt
     3061 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3121 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3181 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     3241 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     3301 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     3361 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     3421 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg
     3481 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     3541 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     3601 atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt
     3661 ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc
     3721 ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct
     3781 ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct
     3841 gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca
     3901 ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg
     3961 gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt
     4021 tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc
     4081 ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt
     4141 tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac
     4201 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4261 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4321 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4381 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4441 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4501 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4561 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     4621 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4681 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt
     4741 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4801 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     4861 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     4921 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4981 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     5041 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     5101 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5161 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5221 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5281 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5341 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5401 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5461 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5521 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5581 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5641 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5701 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5761 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5821 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5881 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5941 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6001 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6061 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6121 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6181 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6241 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6301 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6361 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     6421 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6481 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6541 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6601 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6661 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6721 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6781 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6841 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6901 gctcacatgt
//
