LOCUS       sequence-403527-        7303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flp-dependent expression of Chronos fused to EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7303)
  AUTHORS   Cohen-Kashi Malina K, Tsivourakis E, Kushinsky D, Apelblat D, 
            Shtiglitz S, Zohar E, Sokoletsky M, Tasaka GI, Mizrahi A, Lampl I, 
            Spiegel I
  TITLE     NDNF interneurons in layer 1 gain-modulate whole cortical columns 
            according to an animal's behavioral state.
  JOURNAL   Neuron. 2021 Jul 7;109(13):2150-2164.e5. doi: 
            10.1016/j.neuron.2021.05.001. Epub 2021 May 25.
  PUBMED    34038743
REFERENCE   2  (bases 1 to 7303)
  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"; doi: 
            "10.1016/j.neuron.2021.05.001"; journalName: "Neuron"; date: 
            "2021-07-7- 7"; volume: "109"; issue: "13"; pages: "2150-2164.e5"
COMMENT     Sequence Label: pAAV-Ef1a-fDIO-rc[Chronos-eGFP]-WPRE
FEATURES             Location/Qualifiers
     source          1..7303
                     /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(join(1566..2279,2280..2282))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(1569..2279,2280..2282))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1611..1632)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1960..1979
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2219..2240
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3272..3281
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3288..3321)
                     /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    3430..4018
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3483..3503)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3901..3912)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4050..4526
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4249..4268)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4566..4706
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4566..4695
                     /label=AAV2 ITR
     rep_origin      4781..5236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4868..4887)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5078..5099
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5253..5272)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5372..5394
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5432..5450)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5518..5622
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5623..5691,5692..6483)
                     /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(5841..5860)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6654..7242
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7143..7162
                     /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 gcgccttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
     1621 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc
     1681 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
     1741 tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg
     1801 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
     1861 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     1921 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     1981 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     2041 tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc
     2101 acgagggtgg gccagggcac gggcagcttg ccggtggtgc aaatgaactt cagggtcagc
     2161 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     2221 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     2281 actgctacta ccggtgccgc cactcctccc tcctcctcct cgtcgacgaa ggtctcgatc
     2341 tccatgttct cgccggccac gttgatggtg gttgtcttcc ggatgtcgcc gtggatcagg
     2401 atgtgctcgt ggatcttcac tctcaggtgg tggcccagga agccccaagt gttcttgctg
     2461 atcagatcca ggatcaggtg gccgattccg ctggtgtaag gtgtgatcag gcccagtccc
     2521 tcgtgtccgg cgatgaacat cacggggaac atcagccaag agcagaagaa gacgtaggcc
     2581 atgatcttgc aaatcttccg gcagacgcct ttgggcaggg tgtggtagct ctcgatatac
     2641 accttggcga tctggaagaa gcaagtcacg ccgtagaaca ggccgatcat gaagaacagg
     2701 atcttcaggg ggccctttgt aaaggcggct gtgatccccc acacgatgtt gccgatgtcg
     2761 gtcaccagga tggtcatggt ccgcttgctg tactcttcgt gcagtccggt caggttgctc
     2821 aggtggatca ggatcacggg gcaagtcagg agccacatgc tgtaccgcag ccaaatcacg
     2881 gctcctccgt tggtctggta cactgtggct gggctgtcga cctcgtggaa cagctcgatg
     2941 aagcacttga cgagctcgat cacgcagacg tagacctcct cccagccggt tgtggccttg
     3001 aagctgtgcc aaccgtagaa gatgagctgc acgatagcca cgatcacggt gaaccactgg
     3061 agtcccactg cgatcttgtg ctgcagctcg gttccgtggt tgatgtgggg gaagcaaccg
     3121 tgatcggctc cggctgttgt ggcgttagag gtttctccgt gagcgtctgc tgctggtgtc
     3181 accactgctg cggcgctcag caggcctcta attgtggctt cggcgctagg cacggctgag
     3241 ataaaggcgt gggtcattgt ggcggctgtt tccatggtgg cgctagcgaa gttcctatac
     3301 tttctagaga ataggaactt cttgccttaa cccagaaatt atcactgtta ttctttagaa
     3361 tggtgcaaag agaagttcct ataccttttg aagaatagga acttcgaatt cgatatcaag
     3421 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     3481 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     3541 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     3601 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     3661 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     3721 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     3781 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     3841 tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     3901 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     3961 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     4021 cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc
     4081 ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa
     4141 gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg
     4201 tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac
     4261 caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag
     4321 cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca
     4381 gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca
     4441 actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt
     4501 gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg
     4561 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4621 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4681 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     4741 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     4801 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     4861 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4921 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4981 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5041 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5101 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     5161 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5221 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5281 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5341 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5401 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5461 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5521 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5581 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5641 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5701 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5761 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5821 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5881 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5941 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6001 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6061 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6121 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6181 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6241 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6301 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6361 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6421 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6481 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6541 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6601 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6661 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6721 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6781 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6841 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6901 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6961 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7021 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7081 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7141 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7201 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7261 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
