LOCUS       sequence-298867-        6320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express EGFP from the 
            tetO promoter under intersectional control by Flp and Cre 
            recombinases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6320)
  AUTHORS   Madisen L, Garner AR, Shimaoka D, Chuong AS, Klapoetke NC, Li L, van
            der Bourg A, Niino Y, Egolf L, Monetti C, Gu H, Mills M, Cheng A, 
            Tasic B, Nguyen TN, Sunkin SM, Benucci A, Nagy A, Miyawaki A, 
            Helmchen F, Empson RM, Knopfel T, Boyden ES, Reid RC, Carandini M, 
            Zeng H
  TITLE     Transgenic mice for intersectional targeting of neural sensors and 
            effectors with high specificity and performance.
  JOURNAL   Neuron. 2015 Mar 4;85(5):942-58. doi: 10.1016/j.neuron.2015.02.022.
  PUBMED    25741722
REFERENCE   2  (bases 1 to 6320)
  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.2015.02"; journalName: "Neuron"; date: "2015-03-4-
            4"; volume: "85"; issue: "5"; pages: "942-58"
COMMENT     Sequence Label: AAV pTRE-FSF-FLEX-EGFP-WPRE-bGHpA
FEATURES             Location/Qualifiers
     source          1..6320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    join(287..305,306..328,329..347,348..370,371..389,390..412,
                     413..431,432..454,455..473,474..496,497..515,516..538,
                     539..557)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        590..628
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    750..783
                     /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)"
     polyA_signal    829..877
                     /note="synthetic polyadenylation signal"
     polyA_signal    886..1362
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1085..1104)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(1397..1416)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1441..1489
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    1649..1682
                     /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)"
     protein_bind    complement(1693..1726)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1737..1770)
                     /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)."
     CDS             complement(join(1826..2539,2540..2542,2543..2545))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1829..2539,2540..2542,2543..2545))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1871..1892)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2220..2239
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2479..2500
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2542..2551
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2623..2656
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2667..2700
                     /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)."
     misc_feature    2724..3312
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2777..2797)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3195..3206)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3347..3364)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3353..3560
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3583..3723
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3583..3712
                     /label=AAV2 ITR
     rep_origin      3798..4253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3885..3904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4095..4116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4270..4289)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4389..4411
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4449..4467)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4535..4639
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4640..4708,4709..5500)
                     /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(4858..4877)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5671..6259
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6160..6179
                     /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 gcgaaacaat tctgcaggaa ttcggggccg cggaggctgg
      181 atcggtcccg gtgtcttcta tggaggtcaa aacagcgtgg atggcgtctc caggcgatct
      241 gacggttcac taaacgagct ctgcttatat aggtcgagtt taccactccc tatcagtgat
      301 agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa gtgaaagtcg
      361 agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc
      421 agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga
      481 aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc
      541 cctatcagtg atagagaaaa gtgaaagtcg agctcggtac ccgggtcgag gtaggcgtgt
      601 acggtgggag gcctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg
      661 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccc
      721 cgaattcgag ctcggtaccc ggggacgaag aagttcctat tctctagaaa gtataggaac
      781 ttccctcagc accatggcta gcggcagcct cggagtttga atagatagaa taaaatatct
      841 ttattttcat tccatctgtg tgttggtttt ttgtgtgaga tctacgggtg gcatccctgt
      901 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
      961 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     1021 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     1081 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     1141 gcctcctggg ttcaagcgat tctcctgcct caggctcccg agttgttggg attccaggca
     1201 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     1261 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     1321 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgcctca gctaattgag
     1381 taggggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     1441 cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttca taaacgcggg
     1501 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     1561 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     1621 gcagccaacg tcggggcggc aggccgtgga agttcctatt ctctagaaag tataggaact
     1681 tcctcgacgt cgataacttc gtatagcata cattatacga agttatccga atcgcaataa
     1741 cttcgtatag gatactttat acgaagttat ttggcgcgcc aaatttaaat aacgcaacgt
     1801 accttcgagg gggcccaagg aattcttact tgtacagctc gtccatgccg agagtgatcc
     1861 cggcggcggt cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc
     1921 tcagggcgga ctgggtgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga
     1981 tgggggtgtt ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc
     2041 ggatcttgaa gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt
     2101 ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga
     2161 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg
     2221 tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca
     2281 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca
     2341 cgccgtaggt cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc
     2401 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc
     2461 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga
     2521 acagctcctc gcccttgctc accatggtgg cgaattcccc caagcttgga acgtactaac
     2581 cttgatagct agctagttgg cgcgccaacc tggccggcct tgataacttc gtataatgta
     2641 tgctatacga agttatccga atcgcaataa cttcgtataa agtatcctat acgaagttat
     2701 cgaagatcat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     2761 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     2821 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     2881 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     2941 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     3001 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     3061 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     3121 catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct
     3181 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     3241 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     3301 ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc gatctgcctc gactgtgcct
     3361 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3421 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3481 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3541 aatagcaggc atgctgggga cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg
     3601 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     3661 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     3721 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     3781 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3841 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     3901 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3961 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     4021 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4081 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     4141 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     4201 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     4261 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4321 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4381 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4441 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4501 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4561 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4621 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4681 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4741 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4801 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4861 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4921 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4981 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5041 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5101 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5161 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5221 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5281 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5341 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5401 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5461 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5521 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5581 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5641 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5701 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5761 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5821 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5881 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5941 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6001 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6061 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6121 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6181 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6241 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6301 ctggcctttt gctcacatgt
//
