LOCUS       sequence-329450-        6006 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cre-dependent GCaMP6s under control of the TRE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6006)
  AUTHORS   Sabatini PV, Wang J, Rupp AC, Affinati AH, Flak JN, Li C, Olson DP, 
            Myers MG Jr
  TITLE     tTARGIT AAVs mediate the sensitive and flexible manipulation of 
            intersectional neuronal populations in mice.
  JOURNAL   eLife. 2021 Mar 11;10. pii: 66835. doi: 10.7554/eLife.66835.
  PUBMED    33704065
REFERENCE   2  (bases 1 to 6006)
  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: "eLife."; 
            date: "2021-03-11"; volume: "10. pii: 66835. doi"; pages: " 
            10.7554/eLife.66835"
COMMENT     Sequence Label: pAAV-TRE-DIO-GCaMP6s
FEATURES             Location/Qualifiers
     source          1..6006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   531..671
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   542..671
                     /label=AAV2 ITR
     protein_bind    join(828..846,847..869,870..888,889..911,912..930,931..953,
                     954..972,973..995,996..1014,1015..1037,1038..1056,
                     1057..1079,1080..1098)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        1131..1169
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    complement(1304..1337)
                     /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(1348..1381)
                     /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(1448..1891,1892..1897,1898..2332,2333..2350,
                     2351..2620,2621..2626,2627..2683,2684..2710,2711..2734,
                     2735..2737,2738..2770,2771..2773,2774..2791,2792..2800))
                     /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"
     primer_bind     2007..2026
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2266..2287
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2393..2414)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2695..2704
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2797..2806
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2850..2883
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2894..2927
                     /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    2951..3539
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3004..3024)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3422..3433)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3574..3591)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3580..3787
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3810..3939
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4001..4589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4081..4100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4760..5551,5552..5620))
                     /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     5383..5402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5621..5725)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5793..5811
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5849..5871)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5971..5990
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca atagaccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga
      121 tagagttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg
      481 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc cctgcaggca
      541 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
      601 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      661 taggggttcc tgcggccgca cgcgaaacaa ttctgcagga attcggggcc gcggaggctg
      721 gatcggtccc ggtgtcttct atggaggtca aaacagcgtg gatggcgtct ccaggcgatc
      781 tgacggttca ctaaacgagc tctgcttata taggtcgagt ttaccactcc ctatcagtga
      841 tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc
      901 gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat
      961 cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg
     1021 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact
     1081 ccctatcagt gatagagaaa agtgaaagtc gagctcggta cccgggtcga ggtaggcgtg
     1141 tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac
     1201 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggcc
     1261 ccgaattcga gctcggtacc cggggatcct ctagtcgacg tcgataactt cgtatagcat
     1321 acattatacg aagttatccg aatcgcaata acttcgtata ggatacttta tacgaagtta
     1381 tttggcgcgc caaatttaaa taacgcaacg taccttcgag ggggcccaag gaattcttac
     1441 ttgtacgtca cttcgctgtc atcatttgta caaactcttc gtagtttacc tgaccatccc
     1501 catcgatgtc tgcttccctg atcatttcat caacctcttc atctgttaac ttctctccaa
     1561 ggtttgtcat cacgtggcga agctctgctg cactgatgta gccattgcca tccttatcaa
     1621 acacaccgaa cgcttctcta atttcttctt ccgtgtccct gtatttcatt tttcttgcca
     1681 tcattgtcag gaactcaggg aagtcgattg tgccgtcacc gtcggcatct acttcattga
     1741 tcatgtcctg cagctctgct tctgtggggt tctgccccag agaccgcatc accgtcccca
     1801 gctccttggt tgttattgtc ccatccccgt ccttgtcaaa tagggagaaa gcctctttaa
     1861 attctgcgat ctgctcttca gtcagttggt ccggcaggtt gtactccagc ttgtgcccca
     1921 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt
     1981 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg
     2041 tgcgctcctg gatgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt
     2101 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc
     2161 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg
     2221 catcgccctc accctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct
     2281 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atgctccctc
     2341 cggtaccgcc cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact
     2401 ccagcaggac catgtgatcg cgcttctcgt tggggtcttt cgaaagtttg gactgcacgc
     2461 tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt
     2521 agtggtaggc gagctgcacg ccgccgtcct cgatgttgtg gcggatgtgg aagttcgcct
     2581 tgatgccgtt cttctgcttg tcggccttga tatagacgtt ctcgagtgag ctcagccgac
     2641 ctatagctct gactgcgtga cctgtcttat tccacttacg acgtgatgag tcgaccatgg
     2701 tggcgagatc cttatcgtca tcgtcgtaca gatcccgacc catttgctgt ccaccagtca
     2761 tgctagccat accatgatga tgatgatgat gagaacccat ggtggcggat cctaactagc
     2821 tagttggcgc gccaacctgg ccggccttga taacttcgta taatgtatgc tatacgaagt
     2881 tatccgaatc gcaataactt cgtataaagt atcctatacg aagttatcga agatcatcaa
     2941 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3001 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3061 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3121 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3181 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3241 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3301 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3361 ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3421 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3481 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     3541 tcgataccga gcgctgctcg agagatcgat ctgcctcgac tgtgccttct agttgccagc
     3601 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3661 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3721 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3781 ctggggacac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc
     3841 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     3901 cggcctcagt gagcgagcga gcgcgcagct gcctgcagga catgtgagca aaaggccagc
     3961 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4021 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4081 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4141 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4201 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4261 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4321 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4381 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4441 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4501 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4561 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4621 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4681 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4741 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4801 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4861 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4921 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4981 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5041 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5101 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5161 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5221 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5281 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5341 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5401 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5461 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5521 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5581 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5641 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5701 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     5761 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     5821 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     5881 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     5941 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     6001 accata
//
