LOCUS       sequence-110965-        5400 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express EGFP from the 
            tetO promoter in a Cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5400)
  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 5400)
  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-FLEX-EGFP-WPRE-bGHpA
FEATURES             Location/Qualifiers
     source          1..5400
                     /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
     protein_bind    join(298..316,317..339,340..358,359..381,382..400,401..423,
                     424..442,443..465,466..484,485..507,508..526,527..549,
                     550..568)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        601..638
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    complement(773..806)
                     /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(817..850)
                     /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(906..1619,1620..1622,1623..1625))
                     /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(909..1619,1620..1622,1623..1625))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(951..972)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1300..1319
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1559..1580
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1622..1631
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1703..1736
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1747..1780
                     /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    1804..2392
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1857..1877)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2275..2286)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2427..2444)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2433..2640
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2663..2803
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2663..2792
                     /label=AAV2 ITR
     rep_origin      2878..3333
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2965..2984)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3175..3196
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3350..3369)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3469..3491
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3529..3547)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3615..3719
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3720..3788,3789..4580)
                     /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(3938..3957)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4751..5339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5240..5259
                     /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 cgcgaaacaa ttctgcagga attcggggcc
      181 gcggaggctg gatcggtccc ggtgtcttct atggaggtca aaacagcgtg gatggcgtct
      241 ccaggcgatc tgacggttca ctaaacgagc tctgcttata taggtcgagt ttaccactcc
      301 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
      361 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
      421 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
      481 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga
      541 gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagctcggta cccgggtcga
      601 gtaggcgtgt acggtgggag gcctatataa gcagagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccgcggccc cgaattcgag ctcggtaccc ggggatcctc tagtcgacgt cgataacttc
      781 gtatagcata cattatacga agttatccga atcgcaataa cttcgtatag gatactttat
      841 acgaagttat ttggcgcgcc aaatttaaat aacgcaacgt accttcgagg gggcccaagg
      901 aattcttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
      961 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc
     1021 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
     1081 tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg
     1141 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
     1201 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     1261 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     1321 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     1381 tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc
     1441 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc
     1501 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     1561 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     1621 accatggtgg cgaattcccc caagcttgga acgtactaac cttgatagct agctagttgg
     1681 cgcgccaacc tggccggcct tgataacttc gtataatgta tgctatacga agttatccga
     1741 atcgcaataa cttcgtataa agtatcctat acgaagttat cgaagatcat caagcttatc
     1801 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     1861 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     1921 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     1981 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     2041 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     2101 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     2161 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     2221 ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     2281 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     2341 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     2401 cgagcgctgc tcgagagatc gatctgcctc gactgtgcct tctagttgcc agccatctgt
     2461 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2521 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2581 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2641 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     2701 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     2761 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     2821 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     2881 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2941 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3001 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3061 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     3121 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3181 tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag
     3241 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3301 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     3361 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     3421 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     3481 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     3541 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     3601 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3661 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3721 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3781 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3841 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3901 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3961 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4021 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4081 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4141 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4201 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4261 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4321 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4381 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4441 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4501 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4561 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     4621 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4681 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4741 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4801 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4861 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     4921 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4981 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5041 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5101 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5161 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5221 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5281 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5341 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
