LOCUS       Exported                6916 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  For generating adeno-associated virus to express GFE3 under the EF1a
            promoter in a 'Cre-on' dependent manner..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a-DIO_EGFP-GFE3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6916)
  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 6916)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6916
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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    1450..1483
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    1534..1567
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1767..1847)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(2175..2891)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MGVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELY"
     CDS             complement(2175..2891)
                     /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(2214..2235)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2563..2582
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2822..2843
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(2901..2934)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(2985..3018)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3043..3631
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3096..3116)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3514..3525)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3663..4139
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3862..3881)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4179..4319
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4179..4308
                     /label=AAV2 ITR
     rep_origin      4394..4849
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4481..4500)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4691..4712
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4866..4885)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4985..5007
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5045..5063)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5131..5235
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5236..6096
                     /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(5454..5473)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6267..6855
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6756..6775
                     /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 tcttgaaccg
     1861 gtgcggtagt tgatggagat cgggatcggg tcctggtagt tccagtggtc gatcgtgacg
     1921 gcgtacaccg tgatggtata gtcgacacca ggtttcaggc tgctgatggt agcagtggac
     1981 ttgctgccag gcacggtgaa ttcctggaca gggctattgc caccagtttc accgtaggtg
     2041 atgcggtagt agcgaaccat gaccttgtac ttgccccagc tgatctggat gctggttggt
     2101 gatgcttcct tgacttcgag caattgcgaa cctcctcctg atccacctcc agaacctcca
     2161 ccagaacctc caccgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac
     2221 tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg
     2281 ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg
     2341 tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc
     2401 ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac
     2461 tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg
     2521 cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg
     2581 tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag
     2641 tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg
     2701 gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc
     2761 agcttgccgt aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt
     2821 acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg
     2881 ctcaccccca tatgcctagc ataacttcgt ataaagtatc ctatacgaag ttatttgcct
     2941 taacccagaa attatcactg ttattcttta gaatggtgca aagaataact tcgtataatg
     3001 tatgctatac gaagttatga attcgatatc aagcttatcg ataatcaacc tctggattac
     3061 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3121 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     3181 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3241 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3301 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3361 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3421 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg
     3481 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3541 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3601 agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatct
     3661 acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc
     3721 cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt
     3781 ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga
     3841 caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt
     3901 ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt
     3961 tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac
     4021 ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac
     4081 cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc
     4141 tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt
     4201 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     4261 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     4321 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     4381 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4441 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4501 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4561 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4621 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4681 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat
     4741 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4801 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4861 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4921 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4981 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     5041 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     5101 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5161 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5221 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5281 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5341 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5401 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5461 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5521 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5581 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5641 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5701 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5761 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5821 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5881 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5941 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6001 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6061 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6121 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6181 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6241 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6301 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6361 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     6421 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6481 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6541 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6601 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6661 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6721 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6781 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6841 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6901 ccttttgctc acatgt
//
