LOCUS       sequence-221705-        7314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For cre-dependent 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 7314)
  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 7314)
  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-FLEx-axon-GCaMP6s-P2A-mRuby3
FEATURES             Location/Qualifiers
     source          1..7314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          533..665
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     648..667
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     protein_bind    678..711
                     /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)."
     protein_bind    774..807
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(830..1537)
                     /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"
     CDS             complement(1538..1594)
                     /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             complement(join(1604..2044,2045..2050,2051..2485,2486..2503,
                     2504..2773,2774..2779,2780..2836,2837..2863,2864..2887,
                     2888..2890,2891..2923,2924..2926,2927..2944,2945..2950))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT
                     GHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYETRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSL
                     GQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGNG
                     YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     2419..2440
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2546..2567)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2848..2857
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3046..3079)
                     /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)."
     protein_bind    complement(3150..3183)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3225..3813
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3278..3298)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3696..3707)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3891..4012
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3902..3921)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3956..3975
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4481..4936
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4568..4587)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4778..4799
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4962..5066
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5067..5135,5136..5927)
                     /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(5285..5304)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6098..6686
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6587..6606
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6973..6994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7009..7014,7015..7032,7033..7039)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     repeat_region   7125..7254
                     /label=AAV2 ITR
ORIGIN
        1 gagctccagt gtgctggaat tcgccctttt cagtatttaa attgacgacc gaccccgacc
       61 cactggacaa gcacccaacc cccattcccc aaattgcgca tcccctatca gagaggggga
      121 ggggaaacag gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc
      181 cttcgccccc gcctggcggc gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt
      241 cactcgccgg tcccccgcaa actccccttc ccggccacct tggtcgcgtc cgcgccgccg
      301 ccggcccagc cggaccgcac cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc
      361 tgcgctgcgg cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg
      421 tgtcgtgcct gagagcgcag ctgtgctcct gggcaccgcg cagtccgccc ccgcggctcc
      481 tggccagacc acccctagga ccccctgccc caagtcgcag ccgttggatc aggtaagtat
      541 caaggttaca agacaggttt aaggagacca atagaaactg ggcttgtcga gacagagaag
      601 actcttgcgt ttctgatagg cacctattgg tcttactgac atccactttg cctttctctc
      661 cacaggtgat gatccccata acttcgtata aagtatccta tacgaagtta tatcaaaata
      721 ggaagaccaa tgcttcacca tcgacccgaa ttgccaagca tcaccatcga cacataactt
      781 cgtataatgt atgctatacg aagttatact aggagctcgg cgcgccttac ttgtacagct
      841 cgtccatgcc accaccaagg ttgctgtatt tggcaactgc cacttctctt tgcactacgt
      901 aggtttcatt gtcactctcc tcgatccttt ccaggcggtg atcaacggca tggacaccgg
      961 gcatcttgat gttcccgacg gtcttttttg acctgtaagt tgtcacgaag ttgcagtgca
     1021 gatggccacc accatcaact ttcagtgcga tgtcagtgta tcctctcaga ccaccatctg
     1081 ctggatacat catctctgta ttaggctccc aacccttggt cttcttctgc atcacgggac
     1141 cattggaggg aaagtttacc cctctgacct tgacgttgta gacgagctcg ccatcctcaa
     1201 ggctggtgtc ctgcgtgacg gtgacgactc caccatcttc gtatctcgta actctttccc
     1261 aagtaaaacc ctcaggaaag gactgtttaa agaaatcagg gatgtcggcc gggtacttga
     1321 taaaggtacg gctgccatac atgaacgacg tggcaagaat gtcaaaggca aatggcaggg
     1381 gtcctccctc gatgactttg atcctcatgg tttgcactcc ctcgtacggt ctgccttctc
     1441 cttcacctgt gcatttgaat tggtggccgt tgaccgaacc ttccatgacc accttcatac
     1501 gcatattttc cttgatcagc tcttcgccct tagacacagg accggggttt tcttccacgt
     1561 ctcctgcttg ctttaacaga gagaagttcg tggctccgga tcccttcgct gtcatcattt
     1621 gtacaaactc ttcgtagttt acctgaccat ccccatcgat gtctgcttcc ctgatcattt
     1681 catcaacctc ttcatctgtt aacttctctc caaggtttgt catcacgtgg cgaagctctg
     1741 ctgcactgat gtagccattg ccatccttat caaacacacc gaacgcttct ctaatttctt
     1801 cttccgtgtc cctgtatttc atttttcttg ccatcattgt caggaactca gggaagtcga
     1861 ttgtgccgtc accgtcggca tctacttcat tgatcatgtc ctgcagctct gcttctgtgg
     1921 ggttctgccc cagagaccgc atcaccgtcc ccagctcctt ggttgttatt gtcccatccc
     1981 cgtccttgtc aaatagggag aaagcctctt taaattctgc gatctgctct tcagtcagtt
     2041 ggtccggcag gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga
     2101 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg
     2161 tctcgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggatgtag ccttcgggca
     2221 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca
     2281 cgccgtaggt cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc
     2341 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcaccctcg ccggacacgc
     2401 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga
     2461 acagctcctc gcccttgctc accatgctcc ctccggtacc gcccttgtac agctcgtcca
     2521 tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct
     2581 cgttggggtc tttcgaaagt ttggactgca cgctcaggta gtggttgtcg ggcagcagca
     2641 cggggccgtc gccgatgggg gtgttctgct ggtagtggta ggcgagctgc acgccgccgt
     2701 cctcgatgtt gtggcggatg tggaagttcg ccttgatgcc gttcttctgc ttgtcggcct
     2761 tgatatagac gttctcgagt gagctcagcc gacctatagc tctgactgcg tgacctgtct
     2821 tattccactt acgacgtgat gagtcgacca tggtggcgag atccttatcg tcatcgtcgt
     2881 acagatcccg acccatttgc tgtccaccag tcatgctagc cataccatga tgatgatgat
     2941 gatgagaacc aatcttttgg tcctcatcat tcttttcaac ctgtttggtt cttctcatac
     3001 agcacagcat ggtggcgact agtgctagcg gtaccgaatt ctagtataac ttcgtatagg
     3061 atactttata cgaagttatc attgggattc ttcctatttt gatccaagca tcaccatcga
     3121 ccctctagtc cagatctcac catcgaccca taacttcgta tagcatacat tatacgaagt
     3181 tatgtccctc gaagaggttc gaattcgata tcaagcttat cgataatcaa cctctggatt
     3241 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3301 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3361 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     3421 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     3481 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     3541 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3601 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     3661 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3721 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3781 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgaccc gggcggccgc
     3841 tcgagcatgc atctagagtc gacccgggcg gccgcttcga gcagacatga taagatacat
     3901 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     3961 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     4021 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa
     4081 gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt cctagagcat ggctacgtag
     4141 ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca
     4201 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     4261 cgggctttgc ccgggcggcc tcagtgagcg agcgagggag tggccactcc ctctctgcgc
     4321 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     4381 gcggcctcag tgagcgagcg agcgcgcagc tggcgtaata gcgaagaggc ccgcaccgat
     4441 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     4501 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4561 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4621 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4681 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4741 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4801 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     4861 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4921 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4981 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5041 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5101 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5161 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5221 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5281 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5341 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5401 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5461 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5521 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5581 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5641 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5701 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5761 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     5821 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5881 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     5941 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6001 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6061 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6121 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6181 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6241 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6301 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6361 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6421 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6481 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6541 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6601 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6661 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6721 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6781 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     6841 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     6901 gttggccgat tcattaatgc agctgcgcgc tcgctcgctc aactgggccg cgcgcggtga
     6961 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     7021 gcttccggct cgtatgttgt gtggaattgt gagcggatga cagtgtaaga ccgccaacag
     7081 ctaaraccgc ctctccccgc gcgttggccg attcattaat gcagctgcgc gctcgctcgc
     7141 tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag
     7201 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt
     7261 taatgattaa cccgccatgc tacttatcta cgtagccatg ctctaggaag atcc
//
