LOCUS       sequence-213865-        6789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For enriched expression of GCaMP6s in axons with cytosolic 
            expression of mRuby3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6789)
  AUTHORS   Broussard GJ, Liang Y, Fridman M, Unger EK, Meng G, Xiao X, Ji N, 
            Petreanu L, Tian L
  TITLE     In vivo measurement of afferent activity with axon-specific calcium 
            imaging.
  JOURNAL   Nat Neurosci. 2018 Sep;21(9):1272-1280. doi: 
            10.1038/s41593-018-0211-4. Epub 2018 Aug 20.
  PUBMED    30127424
REFERENCE   2  (bases 1 to 6789)
  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"; doi: 
            "10.1038/s41593-018-0211-4"; journalName: "Nat Neurosci"; date: 
            "2018-09"; volume: "21"; issue: "9"; pages: "1272-1280"
COMMENT     Sequence Label: pAAV-hSynapsin1-axon-GCaMP6s-P2A-mRuby3
FEATURES             Location/Qualifiers
     source          1..6789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   26..155
                     /label=AAV2 ITR
     promoter        237..684
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             join(781..786,787..804,805..807,808..840,841..843,844..867,
                     868..894,895..951,952..957,958..1227,1228..1245,1246..1680,
                     1681..1686,1687..2127)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT
                     GHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSL
                     GQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGNG
                     YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      874..883
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1164..1185
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1291..1312)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1552..1571)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2137..2193
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2194..2901
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTDI
                     ALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAVA
                     KYSNLGGGMDELYK"
     misc_feature    2925..3513
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2978..2998)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3396..3407)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3516..3532)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3517..3533)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3562..3683
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3573..3592)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3627..3646
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4152..4607
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4239..4258)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4449..4470
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4633..4737
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4738..4806,4807..5598)
                     /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(4956..4975)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5769..6357
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6258..6277
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6646..6667
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6682..6687,6688..6705,6706..6712)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 gcgggcctct tcgctattac gccagctgcg cgctcgctcg ctcactgagg ccgcccgggc
       61 aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
      121 agagggagtg gccaactcca tcactagggg ttccttgtag ttaatgatta acccgccatg
      181 ctacttatct acgtagccat gctctaggaa gatctctgca gagggccctg cgtatgagtg
      241 caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac gaccgacccc
      301 gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg
      361 gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca
      421 gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg
      481 acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc
      541 gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac
      601 catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggagga
      661 gtcgtgtcgt gcctgagagc gcagtcgaat tcaagctgct agctagtacc ggtcgccacc
      721 atgctgtgct gtatgagaag aaccaaacag gttgaaaaga atgatgagga ccaaaagatt
      781 ggttctcatc atcatcatca tcatggtatg gctagcatga ctggtggaca gcaaatgggt
      841 cgggatctgt acgacgatga cgataaggat ctcgccacca tggtcgactc atcacgtcgt
      901 aagtggaata agacaggtca cgcagtcaga gctataggtc ggctgagctc actcgagaac
      961 gtctatatca aggccgacaa gcagaagaac ggcatcaagg cgaacttcca catccgccac
     1021 aacatcgagg acggcggcgt gcagctcgcc taccactacc agcagaacac ccccatcggc
     1081 gacggccccg tgctgctgcc cgacaaccac tacctgagcg tgcagtccaa actttcgaaa
     1141 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1201 actctcggca tggacgagct gtacaagggc ggtaccggag ggagcatggt gagcaagggc
     1261 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1321 cacaagttca gcgtgtccgg cgagggtgag ggcgatgcca cctacggcaa gctgaccctg
     1381 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1441 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1501 aagtccgcca tgcccgaagg ctacatccag gagcgcacca tcttcttcaa ggacgacggc
     1561 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1621 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1681 ctgccggacc aactgactga agagcagatc gcagaattta aagaggcttt ctccctattt
     1741 gacaaggacg gggatgggac aataacaacc aaggagctgg ggacggtgat gcggtctctg
     1801 gggcagaacc ccacagaagc agagctgcag gacatgatca atgaagtaga tgccgacggt
     1861 gacggcacaa tcgacttccc tgagttcctg acaatgatgg caagaaaaat gaaatacagg
     1921 gacacggaag aagaaattag agaagcgttc ggtgtgtttg ataaggatgg caatggctac
     1981 atcagtgcag cagagcttcg ccacgtgatg acaaaccttg gagagaagtt aacagatgaa
     2041 gaggttgatg aaatgatcag ggaagcagac atcgatgggg atggtcaggt aaactacgaa
     2101 gagtttgtac aaatgatgac agcgaaggga tccggagcca cgaacttctc tctgttaaag
     2161 caagcaggag acgtggaaga aaaccccggt cctgtgtcta agggcgaaga gctgatcaag
     2221 gaaaatatgc gtatgaaggt ggtcatggaa ggttcggtca acggccacca attcaaatgc
     2281 acaggtgaag gagaaggcag accgtacgag ggagtgcaaa ccatgaggat caaagtcatc
     2341 gagggaggac ccctgccatt tgcctttgac attcttgcca cgtcgttcat gtatggcagc
     2401 cgtaccttta tcaagtaccc ggccgacatc cctgatttct ttaaacagtc ctttcctgag
     2461 ggttttactt gggaaagagt tacgagatac gaagatggtg gagtcgtcac cgtcacgcag
     2521 gacaccagcc ttgaggatgg cgagctcgtc tacaacgtca aggtcagagg ggtaaacttt
     2581 ccctccaatg gtcccgtgat gcagaagaag accaagggtt gggagcctaa tacagagatg
     2641 atgtatccag cagatggtgg tctgagagga tacactgaca tcgcactgaa agttgatggt
     2701 ggtggccatc tgcactgcaa cttcgtgaca acttacaggt caaaaaagac cgtcgggaac
     2761 atcaagatgc ccggtgtcca tgccgttgat caccgcctgg aaaggatcga ggagagtgac
     2821 aatgaaacct acgtagtgca aagagaagtg gcagttgcca aatacagcaa ccttggtggt
     2881 ggcatggacg agctgtacaa gtaagcttac tagagcttat cgataatcaa cctctggatt
     2941 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3001 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3061 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     3121 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     3181 ccacctgtca gctcctttcc gggactttca ctttccccct ccctattgcc acggcggaac
     3241 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3301 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     3361 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3421 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3481 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgacccgggc
     3541 ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     3601 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     3661 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     3721 agggggagat gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg
     3781 ataaggatct tcctagagca tggctacgta gataagtagc atggcgggtt aatcattaac
     3841 tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     3901 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3961 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4021 caaaaggccc ggaaccgtaa acaagtcccg cgccttgccg gtgagcgagc gagcgcgcag
     4081 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4141 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4201 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4261 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4321 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4381 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4441 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4501 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4561 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     4621 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4681 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4741 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4801 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4861 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4921 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4981 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5041 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5101 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5161 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5221 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5281 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5341 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5401 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5461 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5521 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5581 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5641 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5701 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5761 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5821 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5881 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5941 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6001 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6061 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6121 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6181 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6241 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6301 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6361 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6421 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6481 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagcg agcgcgcaga
     6541 gagggagtgg ccaactccat cactaggggc gcgttggccg attcattaat gcagctggca
     6601 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     6661 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     6721 tgtgagcgga taacaggttc acaccggtwc cccattcggg ctgcgcaact gttgggaagg
     6781 gcgatcggt
//
