LOCUS       sequence-231803-        6954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAAV vector for flippase dependent GCaMP6f expression under the 
            control of EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6954)
  AUTHORS   Menegas W, Akiti K, Amo R, Uchida N, Watabe-Uchida M
  TITLE     Dopamine neurons projecting to the posterior striatum reinforce 
            avoidance of threatening stimuli.
  JOURNAL   Nat Neurosci. 2018 Sep 3. pii: 10.1038/s41593-018-0222-1. doi: 
            10.1038/s41593-018-0222-1.
  PUBMED    30177795
REFERENCE   2  (bases 1 to 6954)
  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: "Nat 
            Neurosci. 2018 Sep 3. pii: 10.1038/s41593-018-0222-1. doi: 
            10.1038/s41593-018-0222-1."
COMMENT     Sequence Label: pAAV-EF1a-FLEX(frt)-GCaMP6f-WPRE
FEATURES             Location/Qualifiers
     source          1..6954
                     /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        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1440..1473
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(join(1570..2013,2014..2019,2020..2454,2455..2472,
                     2473..2742,2743..2748,2749..2805,2806..2832,2833..2856,
                     2857..2859,2860..2892,2893..2895,2896..2913,2914..2922))
                     /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"
     primer_bind     2129..2148
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2388..2409
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2515..2536)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2817..2826
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2919..2928
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2939..2972)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    3081..3669
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3134..3154)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3552..3563)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3701..4177
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3900..3919)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4217..4357
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4217..4346
                     /label=AAV2 ITR
     rep_origin      4432..4887
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4519..4538)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4729..4750
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4904..4923)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5023..5045
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5083..5101)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5169..5273
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5274..5342,5343..6134)
                     /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(5492..5511)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6305..6893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6794..6813
                     /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggag
     1441 aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag gaacttcggc
     1561 gcgggccgct cacttcgctg tcatcatttg tacaaactct tcgtagttta cctgaccatc
     1621 cccatcgatg tctgcttccc tgatcatttc atcaacctct tcatctgtta acttctctcc
     1681 aaggtttgtc atcacgtggc gaagctctgc tgcactgatg tagccattgc catccttatc
     1741 aaacacaccg aacgcttctc taatttcttc ttccgtgtcc ctgtatttca tttttcttgc
     1801 catcattgtc aggaactcag ggaagtcgat tgtgccgtca ccgtcggcat ctacttcatt
     1861 gatcatgtcc tgcagctctg cttctgtggg gttctgcccc agagaccgca tcaccgtccc
     1921 cagctccttg gttgttattg tcccatcccc gtccttgtca aatagggaga attcctcttt
     1981 aaattctgcg atctgctctt cagtcagttg gtccggcagg ttgtactcca gcttgtgccc
     2041 caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt
     2101 gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat
     2161 ggtgcgctcc tggatgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat
     2221 gtggtcgggg tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg
     2281 ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt
     2341 ggcatcgccc tcaccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag
     2401 ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatgctccc
     2461 tccggtaccg cccttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa
     2521 ctccagcagg accatgtgat cgcgcttctc gttggggtct ttcgaaagtt tggactgcac
     2581 gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg
     2641 gtagtggtag gcgagctgca cgccgccgtc ctcgatgttg tggcggatct tgaagttcgc
     2701 cttgatgccg ttcttctgct tgtcggcctt gatatagacg ttctcgagtg agctcagccg
     2761 acctatagct ctgactgcgt gacctgtctt attccactta cgacgtgatg agtcgaccat
     2821 ggtggcgaga tccttatcgt catcgtcgta cagatcccga cccatttgct gtccaccagt
     2881 catgctagcc ataccatgat gatgatgatg atgagaaccc atggtggcga gatctagcga
     2941 agttcctata ctttctagag aataggaact tcttgcctta acccagaaat tatcactgtt
     3001 attctttaga atggtgcaaa gagaagttcc tatacctttt gaagaatagg aacttcgaat
     3061 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     3121 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     3181 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     3241 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     3301 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     3361 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     3421 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     3481 cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct
     3541 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     3601 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     3661 cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc
     3721 ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct
     3781 aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg
     3841 gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt
     3901 ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc
     3961 ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat
     4021 gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag
     4081 gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg
     4141 attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg
     4201 cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     4261 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     4321 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     4381 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     4441 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     4501 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     4561 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4621 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     4681 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4741 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     4801 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4861 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     4921 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4981 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     5041 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     5101 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     5161 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5221 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5281 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5341 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5401 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5461 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5521 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5581 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5641 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5701 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5761 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5821 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5881 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5941 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6001 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6061 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6121 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6181 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6241 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6301 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6361 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6421 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6481 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6541 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6601 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6661 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6721 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6781 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6841 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6901 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
