LOCUS       sequence-403530-        7463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flp-dependent Cre and mRuby3 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7463)
  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 7463)
  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-mRuby3-2A-Cre
FEATURES             Location/Qualifiers
     source          1..7463
                     /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(2694..3404)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     CDS             complement(3414..3434)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(3448..3481)
                     /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    3590..4178
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3643..3663)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4061..4072)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4210..4686
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4409..4428)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4726..4866
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4726..4855
                     /label=AAV2 ITR
     rep_origin      4941..5396
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5028..5047)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5238..5259
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5413..5432)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5532..5554
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5592..5610)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5678..5782
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5783..5851,5852..6643)
                     /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(6001..6020)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6814..7402
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7303..7322
                     /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 tgccaccacc aaggttgctg tatttggcaa ctgccacttc tctttgcact
     2761 acgtaggttt cattgtcact ctcctcgatc ctttccaggc ggtgatcaac ggcatggaca
     2821 ccgggcatct tgatgttccc gacggtcttt tttgacctgt aagttgtcac gaagttgcag
     2881 tgcagatggc caccaccatc aactttcagt gcgatgtcag tgtatcctct cagaccacca
     2941 tctgctggat acatcatctc tgtattaggc tcccaaccct tggtcttctt ctgcatcacg
     3001 ggaccattgg agggaaagtt tacccctctg accttgacgt tgtagacgag ctcgccatcc
     3061 tcaaggctgg tgtcctgcgt gacggtgacg actccaccat cttcgtatct cgtaactctt
     3121 tcccaagtaa aaccctcagg aaaggactgt ttaaagaaat cagggatgtc ggccgggtac
     3181 ttgataaagg tacggctgcc atacatgaac gacgtggcaa gaatgtcaaa ggcaaatggc
     3241 aggggtcctc cctcgatgac tttgatcctc atggtttgca ctccctcgta cggtctgcct
     3301 tctccttcac ctgtgcattt gaattggtgg ccgttgaccg aaccttccat gaccaccttc
     3361 atacgcatat tttccttgat cagctcttcg cccttagaca ccatagcggc cgccaccttc
     3421 ctcttcttct tgggcatggt ggctagcgaa gttcctatac tttctagaga ataggaactt
     3481 cttgccttaa cccagaaatt atcactgtta ttctttagaa tggtgcaaag agaagttcct
     3541 ataccttttg aagaatagga acttcgaatt cgatatcaag cttatcgata atcaacctct
     3601 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     3661 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     3721 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     3781 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     3841 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     3901 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     3961 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc
     4021 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     4081 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     4141 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga
     4201 gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
     4261 gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
     4321 taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
     4381 gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
     4441 caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
     4501 cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
     4561 tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
     4621 tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
     4681 gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga
     4741 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     4801 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     4861 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4921 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4981 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     5041 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     5101 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     5161 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5221 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     5281 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     5341 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     5401 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     5461 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5521 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5581 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     5641 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5701 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5761 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5821 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5881 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5941 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     6001 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     6061 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     6121 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6181 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6241 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6301 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6361 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6421 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6481 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6541 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6601 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6661 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6721 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6781 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6841 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6901 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6961 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     7021 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     7081 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     7141 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     7201 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7261 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7321 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7381 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7441 ttgctggcct tttgctcaca tgt
//
