LOCUS       sequence-403523-        7438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flp-dependent Cre and EGFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7438)
  AUTHORS   Roethler O, Zohar E, Cohen-Kashi Malina K, Bitan L, Gabel HW, 
            Spiegel I
  TITLE     Single genomic enhancers drive experience-dependent GABAergic 
            plasticity to maintain sensory processing in the adult cortex.
  JOURNAL   Neuron. 2023 Jun 20:S0896-6273(23)00399-9. doi: 
            10.1016/j.neuron.2023.05.026.
  PUBMED    37354902
REFERENCE   2  (bases 1 to 7438)
  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: "Neuron. 
            2023 Jun 20:S0896-6273(23)00399-9. doi: 
            10.1016/j.neuron.2023.05.026."
COMMENT     Sequence Label: pAAV-Ef1a-fDIO-eGFP-2A-Cre-WPRE
FEATURES             Location/Qualifiers
     source          1..7438
                     /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    1440..1473
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(1566..2594)
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     CDS             complement(2595..2615)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2631..2684)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(join(2694..3404,3405..3407,3408..3410))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2694..3404,3405..3407,3408..3410))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2736..2757)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3085..3104
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      3407..3416
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3423..3456)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    3565..4153
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3618..3638)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4036..4047)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4185..4661
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4384..4403)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4701..4841
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4701..4830
                     /label=AAV2 ITR
     rep_origin      4916..5371
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5003..5022)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5213..5234
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5388..5407)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5507..5529
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5567..5585)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5653..5757
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5758..5826,5827..6618)
                     /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(5976..5995)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6789..7377
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7278..7297
                     /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 gactccggag
     1441 aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag gaacttcggc
     1561 gcgccctaat cgccatcttc cagcaggcgc accattgccc ctgtttcact atccaggtta
     1621 cggatatagt tcatgacaat atttacattg gtccagccac cagcttgcat gatctccggt
     1681 attgaaactc cagcgcgggc catatctcgc gcggctccga cacgggcact gtgtccagac
     1741 caggccaggt atctctgacc agagtcatcc ttagcgccgt aaatcaatcg atgagttgct
     1801 tcaaaaatcc cttccagggc gcgagttgat agctggctgg tggcagatgg cgcggcaaca
     1861 ccattttttc tgacccggca aaacaggtag ttattcggat catcagctac accagagacg
     1921 gaaatccatc gctcgaccag tttagttacc cccaggctaa gtgccttctc tacacctgcg
     1981 gtgctaacca gcgttttcgt tctgccaata tggattaaca ttctcccacc gtcagtacgt
     2041 gagatatctt taaccctgat cctggcaatt tcggctatac gtaacagggt gttataagca
     2101 atccccagaa atgccagatt acgtatatcc tggcagcgat cgctattttc catgagtgaa
     2161 cgaacctggt cgaaatcagt gcgttcgaac gctagagcct gttttgcacg ttcaccggca
     2221 tcaacgtttt cttttcggat ccgccgcata accagtgaaa cagcattgct gtcacttggt
     2281 cgtggcagcc cggaccgacg atgaagcatg tttagctggc ccaaatgttg ctggatagtt
     2341 tttactgcca gaccgcgcgc ctgaagatat agaagataat cgcgaacatc ttcaggttct
     2401 gcgggaaacc atttccggtt attcaacttg caccatgccg cccacgaccg gcaaacggac
     2461 agaagcattt tccaggtatg ctcagaaaac gcctggcgat ccctgaacat gtccatcagg
     2521 ttcttgcgaa cctcatcact cgttgcatcg accggtaatg caggcaaatt ttggtgtacg
     2581 gtcagtaaat tggacacctt cctcttcttc ttgggggttc tgaaagcgct tgggccagga
     2641 ttctcctcga cgtcaccgca tgttagcaga cttcctctgc cctctccact gcccttgtac
     2701 agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga
     2761 tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta gtggttgtcg
     2821 ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc
     2881 acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc
     2941 ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg
     3001 atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg
     3061 ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg
     3121 cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg
     3181 tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag ggtgggccag
     3241 ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca
     3301 tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg
     3361 accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat ggtggcgcta
     3421 gcgaagttcc tatactttct agagaatagg aacttcttgc cttaacccag aaattatcac
     3481 tgttattctt tagaatggtg caaagagaag ttcctatacc ttttgaagaa taggaacttc
     3541 gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     3601 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     3661 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     3721 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     3781 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     3841 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     3901 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     3961 tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc
     4021 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     4081 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     4141 gccgcctccc cgcatcgata ccgagcgctg ctcgagagat ctacgggtgg catccctgtg
     4201 acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg
     4261 tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg
     4321 ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg
     4381 gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg
     4441 cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat
     4501 gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg
     4561 ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc
     4621 tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca
     4681 cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4741 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4801 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     4861 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     4921 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4981 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     5041 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     5101 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     5161 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5221 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     5281 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5341 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     5401 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5461 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5521 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5581 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5641 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5701 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5761 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5821 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5881 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5941 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     6001 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     6061 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     6121 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     6181 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6241 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6301 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6361 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6421 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6481 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6541 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6601 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6661 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6721 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6781 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6841 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6901 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6961 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7021 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7081 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7141 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7201 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7261 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7321 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7381 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
