LOCUS       sequence-201691-        5819 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Amplified expression of GCaMP6f in combination with TET driver.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5819)
  AUTHORS   Sadakane O, Masamizu Y, Watakabe A, Terada S, Ohtsuka M, Takaji M, 
            Mizukami H, Ozawa K, Kawasaki H, Matsuzaki M, Yamamori T
  TITLE     Long-Term Two-Photon Calcium Imaging of Neuronal Populations with 
            Subcellular Resolution in Adult Non-human Primates.
  JOURNAL   Cell Rep. 2015 Dec 1;13(9):1989-99. doi: 
            10.1016/j.celrep.2015.10.050. Epub 2015 Nov 19.
  PUBMED    26655910
REFERENCE   2  (bases 1 to 5819)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2015.10.050"; journalName: "Cell Rep"; date: 
            "2015-12-1- 1"; volume: "13"; issue: "9"; pages: "1989-99"
COMMENT     Sequence Label: pAAVTREGCaMP6f
FEATURES             Location/Qualifiers
     source          1..5819
                     /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        173..551
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    181..199
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    217..235
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    253..271
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    289..307
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    325..343
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    361..379
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    397..415
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     460..484
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      558..567
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(564..572,573..590,591..593,594..626,627..629,630..653,
                     654..680,681..737,738..743,744..1013,1014..1031,1032..1466,
                     1467..1472,1473..1916)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      660..669
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     950..971
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1077..1098)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1338..1357)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    1946..2534
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1999..2019)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2417..2428)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2566..3042
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2765..2784)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3082..3222
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3082..3211
                     /label=AAV2 ITR
     rep_origin      3297..3752
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3384..3403)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3594..3615
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3769..3788)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3888..3910
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3948..3966)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4034..4138
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4139..4207,4208..4999)
                     /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(4357..4376)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5170..5758
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5659..5678
                     /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 gcgtgaggcc ctttcgtctt caagaattcc tcgagtttac
      181 tccctatcag tgatagagaa cgtatgaaga gtttactccc tatcagtgat agagaacgta
      241 tgcagacttt actccctatc agtgatagag aacgtataag gagtttactc cctatcagtg
      301 atagagaacg tatgaccagt ttactcccta tcagtgatag agaacgtatc tacagtttac
      361 tccctatcag tgatagagaa cgtatatcca gtttactccc tatcagtgat agagaacgta
      421 taagctttag gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca
      481 gatcgcctgg agcaattcca caacactttt gtcttatacc aactttccgt accacttcct
      541 accctcgtaa agtcgacgcc accatgggtt ctcatcatca tcatcatcat ggtatggcta
      601 gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatctcg
      661 ccaccatggt cgactcatca cgtcgtaagt ggaataagac aggtcacgca gtcagagcta
      721 taggtcggct gagctcactc gagaacgtct atatcaaggc cgacaagcag aagaacggca
      781 tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc
      841 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
      901 tgagcgtgca gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc
      961 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta
     1021 ccggagggag catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1081 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggtgagggcg
     1141 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1201 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1261 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac atccaggagc
     1321 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1381 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1441 tcctggggca caagctggag tacaacctgc cggaccaact gactgaagag cagatcgcag
     1501 aatttaaaga ggaattctcc ctatttgaca aggacgggga tgggacaata acaaccaagg
     1561 agctggggac ggtgatgcgg tctctggggc agaaccccac agaagcagag ctgcaggaca
     1621 tgatcaatga agtagatgcc gacggtgacg gcacaatcga cttccctgag ttcctgacaa
     1681 tgatggcaag aaaaatgaaa tacagggaca cggaagaaga aattagagaa gcgttcggtg
     1741 tgtttgataa ggatggcaat ggctacatca gtgcagcaga gcttcgccac gtgatgacaa
     1801 accttggaga gaagttaaca gatgaagagg ttgatgaaat gatcagggaa gcagacatcg
     1861 atggggatgg tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg aagtgaggat
     1921 ccaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     1981 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     2041 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     2101 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     2161 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     2221 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     2281 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     2341 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc
     2401 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     2461 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     2521 ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt
     2581 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     2641 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     2701 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     2761 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     2821 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     2881 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     2941 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     3001 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc
     3061 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     3121 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     3181 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     3241 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     3301 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3361 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3421 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3481 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     3541 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3601 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     3661 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3721 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     3781 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     3841 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3901 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     3961 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4021 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4081 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4141 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4201 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4261 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4321 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4381 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4441 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4501 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4561 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4621 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4681 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4741 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4801 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     4861 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4921 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4981 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5041 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5101 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5161 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5221 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5281 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5341 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5401 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5461 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5521 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5581 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5641 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5701 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5761 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
