LOCUS       sequence-210588-        6787 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 6787)
  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 6787)
  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..6787
                     /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"
     repeat_region   3845..3974
                     /label=AAV2 ITR
     primer_bind     complement(3993..4010)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3993..4009)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4002..4024)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4151..4606
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4238..4257)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4448..4469
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4632..4736
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4737..4805,4806..5597)
                     /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(4955..4974)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5768..6356
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6257..6276
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6644..6665
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6680..6685,6686..6703,6704..6710)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6718..6734
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     6723..6745
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6742..6758
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6742..6758
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cgcagaattt aaagaggtaa ttaggctgcg 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 gggactttcg 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 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     3961 gagcgagcgc gcagccttaa ttaacctaat tcactggccg tcgttttaca acgtcgtgac
     4021 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     4081 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     4141 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4201 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4261 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4321 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4381 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4441 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     4501 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4561 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     4621 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4681 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4741 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4801 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     4861 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4921 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4981 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5041 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5101 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5161 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5221 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5281 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5341 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5401 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5461 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5521 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5581 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5641 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5701 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5761 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5821 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5881 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5941 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6001 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6061 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6121 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6181 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6241 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6301 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6361 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6421 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6481 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     6541 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     6601 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     6661 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     6721 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc agatttaatt
     6781 aaggcct
//
