LOCUS       sequence-364317-        5343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of Fluorescent calcium reporter, GCaMP6s from TRE 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5343)
  AUTHORS   Dorgans K, Guo D, Kurima K, Wickens J, Uusisaari MY
  TITLE     Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations
            of Inferior Olive Network in vivo.
  JOURNAL   Front Cell Neurosci. 2022 Apr 27;16:825056. doi: 
            10.3389/fncel.2022.825056. eCollection 2022.
  PUBMED    35573836
REFERENCE   2  (bases 1 to 5343)
  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"; journalName: "Front Cell 
            Neurosci."; date: "2022-04-27"; volume: "16:825056. doi"; pages: " 
            10.3389/fncel.2022.825056. eCollection 2022"
COMMENT     Sequence Label: pAAV-TRE-GCaMP6s-WPRE-sv40
FEATURES             Location/Qualifiers
     source          1..5343
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    18..36
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    54..72
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        84..122
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     119..143
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      185..194
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(191..199,200..217,218..220,221..253,254..256,257..280,
                     281..307,308..364,365..370,371..640,641..658,659..1093,
                     1094..1099,1100..1543)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      287..296
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     577..598
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(704..725)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(965..984)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    1566..2154
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1619..1639)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2037..2048)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2157..2173)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2158..2174)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    2203..2324
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2214..2233)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2268..2287
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(2703..3291)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2783..2802)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3462..4253,4254..4322))
                     /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     4085..4104
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4323..4427)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4453..4908)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4590..4611)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4802..4821
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   4980..5109
                     /label=AAV2 ITR
     protein_bind    5183..5201
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5219..5237
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5254..5272
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5290..5308
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    join(5326..5343,1)
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 aacgtatgtc gagtttatcc ctatcagtga tagagaacgt atgtcgagtt tactccctat
       61 cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg ggaggcctat ataagcagag
      121 ctcgtttagt gaaccgtcag atcgcctgga ggtaccgaat tcaagctgct agctagtacc
      181 ggtcgccacc atgggttctc atcatcatca tcatcatggt atggctagca tgactggtgg
      241 acagcaaatg ggtcgggatc tgtacgacga tgacgataag gatctcgcca ccatggtcga
      301 ctcatcacgt cgtaagtgga ataagacagg tcacgcagtc agagctatag gtcggctgag
      361 ctcactcgag aacgtctata tcaaggccga caagcagaag aacggcatca aggcgaactt
      421 ccacatccgc cacaacatcg aggacggcgg cgtgcagctc gcctaccact accagcagaa
      481 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcgtgcagtc
      541 caaactttcg aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
      601 cgccgccggg atcactctcg gcatggacga gctgtacaag ggcggtaccg gagggagcat
      661 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      721 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg
      781 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      841 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
      901 gcacgacttc ttcaagtccg ccatgcccga aggctacatc caggagcgca ccatcttctt
      961 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1021 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1081 gctggagtac aacctgccgg accaactgac tgaagagcag atcgcagaat ttaaagaggc
     1141 tttctcccta tttgacaagg acggggatgg gacaataaca accaaggagc tggggacggt
     1201 gatgcggtct ctggggcaga accccacaga agcagagctg caggacatga tcaatgaagt
     1261 agatgccgac ggtgacggca caatcgactt ccctgagttc ctgacaatga tggcaagaaa
     1321 aatgaaatac agggacacgg aagaagaaat tagagaagcg ttcggtgtgt ttgataagga
     1381 tggcaatggc tacatcagtg cagcagagct tcgccacgtg atgacaaacc ttggagagaa
     1441 gttaacagat gaagaggttg atgaaatgat cagggaagca gacatcgatg gggatggtca
     1501 ggtaaactac gaagagtttg tacaaatgat gacagcgaag taaaagctta ctagagctta
     1561 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     1621 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     1681 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     1741 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     1801 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc actttccccc
     1861 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     1921 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     1981 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     2041 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     2101 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     2161 accgtcgacc tcgacccggg cggccgcttc gagcagacat gataagatac attgatgagt
     2221 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     2281 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     2341 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc
     2401 tctacaaatg tggtaaaatc gataaggatc ttcctagagc atggctacgt agataagtag
     2461 catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct
     2521 ctgcgcgctc gctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2581 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2641 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2701 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2761 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2821 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2881 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2941 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3001 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3061 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3121 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3181 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3241 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3301 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3361 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3421 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3481 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3541 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3601 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3661 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3721 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3781 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3841 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3901 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3961 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4021 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4081 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4141 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4201 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4261 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4321 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4381 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4441 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     4501 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     4561 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     4621 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     4681 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     4741 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     4801 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     4861 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
     4921 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     4981 tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg
     5041 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
     5101 ggggttcctt gtagttaatg attaacccgc catgctactt atctacgtag ccatgctcta
     5161 ggaagatctc gaattggttt actccctatc agtgatagag aacgtatgtc gagtttactc
     5221 cctatcagtg atagagaacg atgtcgagtt tactccctat cagtgataga gaacgtatgt
     5281 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
     5341 gag
//
