LOCUS       sequence-50207-e        6470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP in cells lacking Cre (Cre-Off).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6470)
  AUTHORS   Saunders A, Johnson CA, Sabatini BL
  TITLE     Novel recombinant adeno-associated viruses for Cre activated and 
            inactivated transgene expression in neurons.
  JOURNAL   Front Neural Circuits. 2012 Jul 27;6:47. doi: 
            10.3389/fncir.2012.00047. eCollection 2012.
  PUBMED    22866029
REFERENCE   2  (bases 1 to 6470)
  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: "Front 
            Neural Circuits."; date: "2012-07-27"; volume: "6:47. doi"; pages: "
            10.3389/fncir.2012.00047. eCollection 2012"
COMMENT     Sequence Label: pAAV-Ef1a-DO-EGFP-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6470
                     /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)."
     regulatory      1589..1598
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1595..1597,1598..1600,1601..2311,2312..2317,
                     2318..2440)
                     /codon_start=1
                     /product="EGFP destabilized by fusion to a mutated version 
                     of residues 422-461 of mouse ornithine decarboxylase, 
                     giving an in vivo half-life of ~1 hour"
                     /label=d1EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYKKLSHGFPPAVAAQDDGTLPMSCAQESGMDRHPAACASARINV
                     "
     primer_bind     complement(1640..1661)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1901..1920)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2248..2269
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2455..2488
                     /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    2539..2572
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2597..3185
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2650..2670)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3068..3079)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3217..3693
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3416..3435)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3733..3873
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3733..3862
                     /label=AAV2 ITR
     rep_origin      3948..4403
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4035..4054)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4245..4266
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4420..4439)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4539..4561
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4599..4617)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4685..4789
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4790..4858,4859..5650)
                     /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(5008..5027)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5821..6409
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6310..6329
                     /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 gcgccttatc tagatttcgc caccatggtg agcaagggcg aggagctgtt
     1621 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1681 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1741 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1801 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1861 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1921 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1981 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     2041 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     2101 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2161 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     2221 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2281 gatcactctc ggcatggacg agctgtacaa gaagcttagc catggcttcc cgccggcggt
     2341 ggcggcgcag gatgatggca cgctgcccat gtcttgtgcc caggagagcg ggatggaccg
     2401 tcaccctgca gcctgtgctt ctgctaggat caatgtgtag gcggccgcgc tagcataact
     2461 tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc
     2521 tttagaatgg tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga
     2581 tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     2641 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     2701 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     2761 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     2821 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     2881 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     2941 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     3001 ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta
     3061 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     3121 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     3181 cccgcatcga taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc
     3241 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
     3301 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
     3361 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat
     3421 tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg
     3481 gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc
     3541 aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg
     3601 gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta
     3661 caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga
     3721 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     3781 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     3841 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     3901 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     3961 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4021 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4081 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4141 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     4201 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4261 aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc
     4321 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4381 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     4441 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4501 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4561 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     4621 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     4681 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4741 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4801 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4861 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4921 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4981 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5041 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5101 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5161 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     5221 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     5281 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     5341 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     5401 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5461 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     5521 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5581 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5641 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     5701 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5761 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5821 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5881 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5941 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     6001 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6061 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6121 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6181 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6241 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6301 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6361 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6421 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
