LOCUS       sequence-210581-        6772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For enriched expression of GCaMP6s in axons with cytosolic 
            expression of mKate2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6772)
  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 6772)
  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-mKate2
FEATURES             Location/Qualifiers
     source          1..6772
                     /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..2889
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     misc_feature    2910..3498
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2963..2983)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3381..3392)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3501..3517)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3502..3518)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3547..3668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3558..3577)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3612..3631
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3830..3959
                     /label=AAV2 ITR
     primer_bind     complement(3978..3995)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3978..3994)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3987..4009)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4136..4591
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4223..4242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4433..4454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4617..4721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4722..4790,4791..5582)
                     /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(4940..4959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5753..6341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6242..6261
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6629..6650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6665..6670,6671..6688,6689..6695)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6703..6719
                     /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     6708..6730
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6727..6743
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6727..6743
                     /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 cctgtgagcg agctgattaa ggagaacatg
     2221 cacatgaagc tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag
     2281 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggcggt cgagggcggc
     2341 cctctcccct tcgccttcga catcctggct accagcttca tgtacggcag caaaaccttc
     2401 atcaaccaca cccagggcat ccccgacttc tttaagcagt ccttccccga gggcttcaca
     2461 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
     2521 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac
     2581 ggccctgtga tgcagaagaa aacactcggc tgggaggcct ccaccgagac cctgtacccc
     2641 gctgacggcg gcctggaagg cagagccgac atggccctga agctcgtggg cgggggccac
     2701 ctgatctgca acttgaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg
     2761 cccggcgtct actatgtgga cagaagactg gaaagaatca aggaggccga caaagagacc
     2821 tacgtcgagc agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg
     2881 cacagataag cttactagag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     2941 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3001 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     3061 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     3121 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     3181 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     3241 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     3301 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     3361 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     3421 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     3481 tttgggccgc ctccccgcat cgataccgtc gacctcgacc cgggcggccg cttcgagcag
     3541 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     3601 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     3661 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg
     3721 aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatcttccta
     3781 gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta
     3841 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     3901 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     3961 cttaattaac ctaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     4021 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     4081 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     4141 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4201 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4261 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4321 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     4381 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4441 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     4501 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4561 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     4621 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4681 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4741 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4801 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4861 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4921 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4981 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5041 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5101 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5161 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5221 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5281 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5341 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5401 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5461 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5521 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5581 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5641 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5701 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5761 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5821 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5881 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5941 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6001 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6061 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6121 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6181 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6241 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6301 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6361 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     6421 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     6481 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     6541 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     6601 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     6661 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     6721 ttcacacagg aaacagctat gaccatgatt acgccagatt taattaaggc ct
//
