LOCUS       sequence-110998-        6613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express the tetracycline
            transactivator tTA2 from the CAG promoter in a Cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6613)
  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 6613)
  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 pCAG-FLEX2-tTA2-WPRE-bGHpA
FEATURES             Location/Qualifiers
     source          1..6613
                     /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
     enhancer        234..537
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        539..816
                     /label=chicken beta-actin promoter
     intron          818..1835
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1759..1781
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1843..1862
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1928..1937
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1940..1973
                     /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)."
     protein_bind    2036..2069
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(2086..2094,2095..2130,2131..2133,2134..2169,
                     2170..2172,2173..2208,2209..2214,2215..2826))
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="RLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKN
                     KRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPT
                     EKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDS
                     MPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPADA
                     LDDFDLDMLPADALDDFDLDMLPG"
     protein_bind    complement(2838..2871)
                     /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)."
     protein_bind    complement(2942..2975)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2981..2990
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    3017..3605
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3070..3090)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3488..3499)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3640..3657)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3646..3853
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3876..4016
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3876..4005
                     /label=AAV2 ITR
     rep_origin      4091..4546
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4178..4197)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4388..4409
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4563..4582)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4682..4704
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4742..4760)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4828..4932
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4933..5001,5002..5793)
                     /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(5151..5170)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5964..6552
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6453..6472
                     /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 atctagttat
      181 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      241 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      301 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      361 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      421 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      481 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      541 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      601 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      661 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      721 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      781 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggga gtcgctgcga cgctgccttc
      841 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
      901 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg
      961 tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg
     1021 gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg
     1081 ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt
     1141 gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg
     1201 ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg
     1261 gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga gttgctgagc
     1321 acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg
     1381 gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg
     1441 gctcggggga ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc
     1501 gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa
     1561 tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga
     1621 agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc
     1681 gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg
     1741 ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg
     1801 ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg
     1861 tgctgtctca tcattttggc aaagaattga tttgataccg cgggccccgc gtggacggta
     1921 ccggcgcgcc accatggcca taacttcgta taaagtatcc tatacgaagt tatatcaaaa
     1981 taggaagacc aatgcttcac catcgacccg aattgccaag catcaccatc gacccataac
     2041 ttcgtataat gtatgctata cgaagttatg tttaaactta attaatcatc cggggagcat
     2101 gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata tcaaggtcaa agtcgtcaag
     2161 ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg gcgtcggccg gcccgccgct
     2221 ttcgcacttt agctgtttct ccaggccaca tatgattagt tccaggccga aaaggaaggc
     2281 aggttcggct ccctgatggt cgaacagctc aattgcttgt ctcagaagtg ggggcataga
     2341 atcggtggta ggtgtctctc tttcctcttt tgctacttga tgctcctgat cctccaatac
     2401 gcagcccagt gtaaagtggc ccacggcgga cagagcgtac agtgcgttct ccagggagaa
     2461 gccttgctga cacaggaacg cgagctgatt ttccagggtt tcgtactgtt tctctgttgg
     2521 gcgggtgccg agatgcactt tagccccgtc gcgatgtgag aggagagcac agcggaatga
     2581 cttggcgttg ttccgcagaa agtcttgcca tgactcgcct tccagggggc agaagtgggt
     2641 atgatgcctg tccagcatct cgattgccag ggcatcgagc agggcccgct tgttcttcac
     2701 gtgccagtac agggtaggct gctcaactcc cagcttttga gcgagtttcc ttgtcgtcag
     2761 gccttcgata ccgacttcat tgagtaattc cagagcagag tttatgactt tgctcttgtc
     2821 cagtctagca ctagtcgata acttcgtata ggatacttta tacgaagtta tctttgggat
     2881 tcttcctatt ttgatccaag catcaccatc gaccctctag tccagatttc accatcgacc
     2941 cataacttcg tatagcatac attatacgaa gttatgatat ccatggtggc gggatatcag
     3001 atctaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     3061 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     3121 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     3181 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     3241 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     3301 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     3361 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     3421 ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta
     3481 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     3541 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     3601 cccgcatcga taccgagcgc tgctcgagag atcgatctgc ctcgactgtg ccttctagtt
     3661 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3721 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3781 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     3841 ggcatgctgg ggacacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc
     3901 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     3961 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     4021 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4081 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4141 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4201 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4261 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4321 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4381 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct
     4441 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4501 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     4561 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4621 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4681 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     4741 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4801 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4861 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4921 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4981 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5041 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5101 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5161 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5221 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5281 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5341 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5401 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5461 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5521 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5581 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5641 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5701 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5761 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5821 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5881 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5941 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6001 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6061 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     6121 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6181 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6241 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6301 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6361 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6421 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6481 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6541 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6601 tttgctcaca tgt
//
