LOCUS       sequence-381181-        5933 bp    DNA     circular SYN 14-APR-2025
DEFINITION  AAV-mediated expression of ultrasensitive protein calcium sensor 
            under the Syn promoter, Cre mediated flip-excision switch.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5933)
  AUTHORS   Dana H, Sun Y, Mohar B, Hulse BK, Kerlin AM, Hasseman JP, Tsegaye G,
            Tsang A, Wong A, Patel R, Macklin JJ, Chen Y, Konnerth A, Jayaraman 
            V, Looger LL, Schreiter ER, Svoboda K, Kim DS
  TITLE     High-performance calcium sensors for imaging activity in neuronal 
            populations and microcompartments.
  JOURNAL   Nat Methods. 2019 Jul;16(7):649-657. doi: 10.1038/s41592-019-0435-6.
            Epub 2019 Jun 17.
  PUBMED    31209382
REFERENCE   2  (bases 1 to 5933)
  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: 
            "10.1038/s41592-019-0435-6"; journalName: "Nat Methods"; date: 
            "2019-07"; volume: "16"; issue: "7"; pages: "649-657"
COMMENT     Sequence Label: pGP-AAV-syn-FLEX-jGCaMP7f-WPRE
FEATURES             Location/Qualifiers
     source          1..5933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(24..471)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   553..682
                     /label=AAV2 ITR
     rep_origin      754..1209
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(841..860)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1051..1072
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1235..1339
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1340..1408,1409..2200)
                     /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(1558..1577)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2371..2959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2860..2879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   3196..3325
                     /label=AAV2 ITR
     polyA_signal    3487..3608
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3524..3543)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3578..3597
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    3651..4239
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3757..3768
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4166..4186
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    4281..4314
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4385..4418
                     /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)."
     regulatory      4429..4438
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4435..4443,4444..4461,4462..4464,4465..4497,4498..4500,
                     4501..4524,4525..4551,4552..4608,4609..4614,4615..4884,
                     4885..4902,4903..5337,5338..5343,5344..5787)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      4531..4540
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     4821..4842
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4948..4969)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5209..5228)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(5798..5831)
                     /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(5894..5927)
                     /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)."
ORIGIN
        1 ccttgctagc agcttgaatt cgactgcgct ctcaggcacg acacgactcc tccgctgccc
       61 accgcagact gaggcagcgc tgagtcgccg gcgccgcagc gcagatggtc gcgcccgtgc
      121 ccccctatct cgcgcctcgc gtggtgcggt ccggctgggc cggcggcggc gcggacgcga
      181 ccaaggtggc cgggaagggg agtttgcggg ggaccggcga gtgacgtcag cgcgccttca
      241 gtgctgaggc ggcggtggcg cgcgccgcca ggcgggggcg aaggcactgt ccgcggtgct
      301 gaagctggca gtgcgcacgc gcctcgccgc atcctgtttc ccctccccct ctctgatagg
      361 ggatgcgcaa tttggggaat gggggttggg tgcttgtcca gtgggtcggg gtcggtcgtc
      421 aggtaggcac ccccaccccg cctcatcctg gtcctaaaac ccacttgcac tcatacgcag
      481 ggccctctgc agagatcttc ctagagcatg gctacgtaga taagtagcat ggcgggttaa
      541 tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
      601 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
      661 cagtgagcga gcgagcgcgc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
      721 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
      781 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      841 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      901 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      961 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1021 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1081 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1141 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1201 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1261 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     1321 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     1381 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     1441 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     1501 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     1561 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     1621 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     1681 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     1741 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     1801 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     1861 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     1921 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     1981 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     2041 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     2101 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     2161 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     2221 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     2281 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2341 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2401 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2461 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     2521 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2581 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2641 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2701 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2761 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     2821 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2881 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     2941 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3001 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3061 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3121 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     3181 gattcattaa tgcagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg
     3241 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg
     3301 gccaactcca tcactagggg ttccttgtag ttaatgatta acccgccatg ctacttatct
     3361 acgtagccat gctctaggaa gatccttatc gattttacca catttgtaga ggttttactt
     3421 gctttaaaaa acctcccaca tctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     3481 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3541 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3601 gtatcttatc atgtctgctc gaagcggccg cccgggtcga cggtatcgat gcggggaggc
     3661 ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa gaggccgcag
     3721 agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag ggacgtagca
     3781 gaaggacgtc ccgcgcagaa tccaggtggc aacacaggcg agcagccaag gaaaggacga
     3841 tgatttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc ctgtccagca
     3901 gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga aagcgaaagt
     3961 cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg cgtcagcaaa
     4021 cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat aaagagacag
     4081 caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa tagcatgata
     4141 caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt taagaatacc
     4201 agtcaatctt tcacaaattt tgtaatccag aggttgatta tcgataagct tgatatcgaa
     4261 ttcgaacctc ttcgagggac ataacttcgt ataatgtatg ctatacgaag ttatgggtcg
     4321 atggtgagat ctggactaga gggtcgatgg tgatgcttgg atcaaaatag gaagaatccc
     4381 aatgataact tcgtataaag tatcctatac gaagttatac taggatccgc caccatgggt
     4441 tctcatcatc atcatcatca tggtatggct agcatgactg gtggacagca aatgggtcgg
     4501 gatctgtacg acgatgacga taaggatctc gccaccatgg tcgactcatc acgtcgtaag
     4561 tggaataaga caggtcacgc agtcagagct ataggtcggc tgagctcact cgagaacgtc
     4621 tatatcaagg ccgacaagca gaagaacggc atcaaggcga acttcaagat ccgccacaac
     4681 atcgaggacg gcggcgtgca gctcgcctac cactaccagc agaacacccc catcggcgac
     4741 ggccccgtgc tgctgcccga caaccactac ctgagcgtgc agtccaaact ttcgaaagac
     4801 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     4861 ctcggcatgg acgagctgta caagggcggt accggaggga gcatggtgag caagggcgag
     4921 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     4981 aagttcagcg tgtccggcga gggtgagggc gatgccacct acggcaagct gaccctgaag
     5041 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     5101 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     5161 tccgccatgc ccgaaggcta catccaggag cgcaccatct tcttcaagga cgacggcaac
     5221 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctt
     5281 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacctg
     5341 ccggaccaac tgactgaaga gcagatcgca gaatttaaag agctgttctc cctatttgac
     5401 aaggacgggg atgggacaat aacaaccaag gagctgggga cggtgatgcg gtctctgggg
     5461 cagaacccca cagaagcaga gctgcaggac atgatcaatg aagtagatgc cgacggtgac
     5521 ggcacaatcg acttccctga gttcctgaca atgatggcaa gaaaaatgaa atacacagac
     5581 agtgaagaag aaattagaga agcgttccgt gtgtttgata aggatggcaa tggctacatc
     5641 agtgcagcag agcttcgcca cgtgatgaca aaccttggag agaagttaac agatgaagag
     5701 gttgatgaaa tgatcaggga agcagacatc gatggggatg gtcaggtaaa ctacgaagag
     5761 tttgtacaaa tgatgacagc gaagtgaaag ctctagtata acttcgtata gcatacatta
     5821 tacgaagtta tgggtcgatg gtgatgcttg gcaattcggg tcgatggtga agcattggtc
     5881 ttcctatttt gatataactt cgtataggat actttatacg aagttatggg gat
//
