LOCUS       sequence-210596-        6730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For co-expression of GCaMP6s with mRuby3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6730)
  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 6730)
  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-GCaMP6s-P2A-mRuby3
FEATURES             Location/Qualifiers
     source          1..6730
                     /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)"
     regulatory      715..724
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(721..729,730..747,748..750,751..783,784..786,787..810,
                     811..837,838..894,895..900,901..1170,1171..1188,1189..1623,
                     1624..1629,1630..2070)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      817..826
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1107..1128
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1234..1255)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1495..1514)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2080..2136
                     /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             2137..2844
                     /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    2868..3456
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2921..2941)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3339..3350)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3459..3475)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3460..3476)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3505..3626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3516..3535)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3570..3589
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3788..3917
                     /label=AAV2 ITR
     primer_bind     complement(3936..3953)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3936..3952)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3945..3967)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4094..4549
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4181..4200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4391..4412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4575..4679
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4680..4748,4749..5540)
                     /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(4898..4917)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5711..6299
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6200..6219
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6587..6608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6623..6628,6629..6646,6647..6653)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6661..6677
                     /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     6666..6688
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6685..6701
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6685..6701
                     /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 atgggttctc atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg
      781 ggtcgggatc tgtacgacga tgacgataag gatctcgcca ccatggtcga ctcatcacgt
      841 cgtaagtgga ataagacagg tcacgcagtc agagctatag gtcggctgag ctcactcgag
      901 aacgtctata tcaaggccga caagcagaag aacggcatca aggcgaactt ccacatccgc
      961 cacaacatcg aggacggcgg cgtgcagctc gcctaccact accagcagaa cacccccatc
     1021 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcgtgcagtc caaactttcg
     1081 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1141 atcactctcg gcatggacga gctgtacaag ggcggtaccg gagggagcat ggtgagcaag
     1201 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1261 ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg caagctgacc
     1321 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1381 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1441 ttcaagtccg ccatgcccga aggctacatc caggagcgca ccatcttctt caaggacgac
     1501 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1561 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1621 aacctgccgg accaactgac tgaagagcag atcgcagaat ttaaagaggc tttctcccta
     1681 tttgacaagg acggggatgg gacaataaca accaaggagc tggggacggt gatgcggtct
     1741 ctggggcaga accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac
     1801 ggtgacggca caatcgactt ccctgagttc ctgacaatga tggcaagaaa aatgaaatac
     1861 agggacacgg aagaagaaat tagagaagcg ttcggtgtgt ttgataagga tggcaatggc
     1921 tacatcagtg cagcagagct tcgccacgtg atgacaaacc ttggagagaa gttaacagat
     1981 gaagaggttg atgaaatgat cagggaagca gacatcgatg gggatggtca ggtaaactac
     2041 gaagagtttg tacaaatgat gacagcgaag ggatccggag ccacgaactt ctctctgtta
     2101 aagcaagcag gagacgtgga agaaaacccc ggtcctgtgt ctaagggcga agagctgatc
     2161 aaggaaaata tgcgtatgaa ggtggtcatg gaaggttcgg tcaacggcca ccaattcaaa
     2221 tgcacaggtg aaggagaagg cagaccgtac gagggagtgc aaaccatgag gatcaaagtc
     2281 atcgagggag gacccctgcc atttgccttt gacattcttg ccacgtcgtt catgtatggc
     2341 agccgtacct ttatcaagta cccggccgac atccctgatt tctttaaaca gtcctttcct
     2401 gagggtttta cttgggaaag agttacgaga tacgaagatg gtggagtcgt caccgtcacg
     2461 caggacacca gccttgagga tggcgagctc gtctacaacg tcaaggtcag aggggtaaac
     2521 tttccctcca atggtcccgt gatgcagaag aagaccaagg gttgggagcc taatacagag
     2581 atgatgtatc cagcagatgg tggtctgaga ggatacactg acatcgcact gaaagttgat
     2641 ggtggtggcc atctgcactg caacttcgtg acaacttaca ggtcaaaaaa gaccgtcggg
     2701 aacatcaaga tgcccggtgt ccatgccgtt gatcaccgcc tggaaaggat cgaggagagt
     2761 gacaatgaaa cctacgtagt gcaaagagaa gtggcagttg ccaaatacag caaccttggt
     2821 ggtggcatgg acgagctgta caagtaagct tactagagct tatcgataat caacctctgg
     2881 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     2941 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3001 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     3061 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     3121 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     3181 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     3241 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     3301 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     3361 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     3421 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgacccg
     3481 ggcggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta
     3541 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     3601 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     3661 ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa
     3721 tcgataagga tcttcctaga gcatggctac gtagataagt agcatggcgg gttaatcatt
     3781 aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     3841 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     3901 agcgagcgag cgcgcagcct taattaacct aattcactgg ccgtcgtttt acaacgtcgt
     3961 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     4021 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     4081 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4141 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4201 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4261 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4321 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4381 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4441 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4501 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     4561 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4621 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4681 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4741 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4801 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4861 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4921 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4981 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5041 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5101 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5161 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5221 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5281 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5341 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5401 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5461 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5521 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5581 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5641 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5701 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5761 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5821 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5881 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5941 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6001 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6061 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6121 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6181 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6241 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6301 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     6361 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     6421 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     6481 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     6541 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     6601 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     6661 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
     6721 attaaggcct
//
