LOCUS       sequence-207278-        7305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In the presence of Cre, can be used to express both GCaMP6s fused to
            the presynaptic protein synaptophysin and independently the 
            red-fluorescent protein mRuby3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7305)
  TITLE     AAV GCaMP6 Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7305)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: AAV_CWSL.nEF.DIO.Synaptophysin-GCaMP6s.P2A.mRuby3
FEATURES             Location/Qualifiers
     source          1..7305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        33..244
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             257..525
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    complement(565..598)
                     /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(649..682)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(686..1393)
                     /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(1394..1450)
                     /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(1460..1900,1901..1906,1907..2341,2342..2359,
                     2360..2629,2630..2635,2636..2692,2693..2719,2720..2743,
                     2744..2746,2747..2779,2780..2782,2783..2800,2801..2809))
                     /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"
     primer_bind     2016..2035
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2275..2296
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2402..2423)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2704..2713
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(join(2820..2834,2835..2843))
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     protein_bind    3815..3848
                     /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    3899..3932
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4019..4039)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4399..4539
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4399..4528
                     /label=AAV2 ITR
     rep_origin      4614..5069
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4701..4720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4911..4932
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5086..5105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5205..5227
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5265..5283)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5351..5455
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5456..5524,5525..6316)
                     /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(5674..5693)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6487..7075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6976..6995
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7137..7277
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7148..7277
                     /label=AAV2 ITR
ORIGIN
        1 aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
       61 cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt
      121 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
      181 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
      241 acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg
      301 ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac
      361 tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct
      421 cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca
      481 actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc tgtgaccggc
      541 gcctactcta gagtcgactc cggaataact tcgtatagga tactttatac gaagttatgc
      601 agaatggtag ctggattgta gctgctatta gcaatatgaa acctcttaat aacttcgtat
      661 agcatacatt atacgaagtt attcacttgt acagctcgtc catgccacca ccaaggttgc
      721 tgtatttggc aactgccact tctctttgca ctacgtaggt ttcattgtca ctctcctcga
      781 tcctttccag gcggtgatca acggcatgga caccgggcat cttgatgttc ccgacggtct
      841 tttttgacct gtaagttgtc acgaagttgc agtgcagatg gccaccacca tcaactttca
      901 gtgcgatgtc agtgtatcct ctcagaccac catctgctgg atacatcatc tctgtattag
      961 gctcccaacc cttggtcttc ttctgcatca cgggaccatt ggagggaaag tttacccctc
     1021 tgaccttgac gttgtagacg agctcgccat cctcaaggct ggtgtcctgc gtgacggtga
     1081 cgactccacc atcttcgtat ctcgtaactc tttcccaagt aaaaccctca ggaaaggact
     1141 gtttaaagaa atcagggatg tcggccgggt acttgataaa ggtacggctg ccatacatga
     1201 acgacgtggc aagaatgtca aaggcaaatg gcaggggtcc tccctcgatg actttgatcc
     1261 tcatggtttg cactccctcg tacggtctgc cttctccttc acctgtgcat ttgaattggt
     1321 ggccgttgac cgaaccttcc atgaccacct tcatacgcat attttccttg atcagctctt
     1381 cgcccttaga cactggcccg ggattctctt cgacatcccc tgcttgtttc aacagggaga
     1441 agttagtggc tccgcttccc ttcgctgtca tcatttgtac aaactcttcg tagtttacct
     1501 gaccatcccc atcgatgtct gcttccctga tcatttcatc aacctcttca tctgttaact
     1561 tctctccaag gtttgtcatc acgtggcgaa gctctgctgc actgatgtag ccattgccat
     1621 ccttatcaaa cacaccgaac gcttctctaa tttcttcttc cgtgtccctg tatttcattt
     1681 ttcttgccat cattgtcagg aactcaggga agtcgattgt gccgtcaccg tcggcatcta
     1741 cttcattgat catgtcctgc agctctgctt ctgtggggtt ctgccccaga gaccgcatca
     1801 ccgtccccag ctccttggtt gttattgtcc catccccgtc cttgtcaaat agggagaaag
     1861 cctctttaaa ttctgcgatc tgctcttcag tcagttggtc cggcaggttg tactccagct
     1921 tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca
     1981 ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga
     2041 agaagatggt gcgctcctgg atgtagcctt cgggcatggc ggacttgaag aagtcgtgct
     2101 gcttcatgtg gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga
     2161 gggtgggcca gggcacgggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc
     2221 cgtaggtggc atcgccctca ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc
     2281 cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca
     2341 tgctccctcc ggtaccgccc ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg
     2401 tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttc gaaagtttgg
     2461 actgcacgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt
     2521 tctgctggta gtggtaggcg agctgcacgc cgccgtcctc gatgttgtgg cggatgtgga
     2581 agttcgcctt gatgccgttc ttctgcttgt cggccttgat atagacgttc tcgagtgagc
     2641 tcagccgacc tatagctctg actgcgtgac ctgtcttatt ccacttacga cgtgatgagt
     2701 cgaccatggt ggcgagatcc ttatcgtcat cgtcgtacag atcccgaccc atttgctgtc
     2761 caccagtcat gctagccata ccatgatgat gatgatgatg agaacccatt ggcgagatcc
     2821 ttatcgtcat cgtcgtacag atcccgaccc atttgctgtc caccagtccc ggtggatcct
     2881 gattggagaa ggaggtgggc gcaccctgtt ggccatagcc ttgctgccca tagtcgccct
     2941 gaggcccgta gccaccgcca ccgctggctg gctgcccgta atcgggttga taaccaccct
     3001 gaggcccgta ggagtcctgg ggcccatagc ctccggggcc ctgcccgtag cccgcatcgc
     3061 cgtaggcatc gccaggtgct ggttgctttt ccggggcgcc tggaggtgcg cgcatgaatg
     3121 gggctgccca gcctgtctcc ttgaacacga accataagtt gccaacccag agcaccaggt
     3181 tcaggaagcc aaacaccact gtggtgttga gtcctgaagt cacagggtcc ctcagttcct
     3241 tgcatgtgtt ccctgtctgg cggcacatgg gcatctcctt gataatgttc tctgggtccg
     3301 tggccatctt cacatcggac aggcctttgg cccaggctga tgaactaact agccacatga
     3361 aagcgaacac ggctgtagcc agaaagtcca tcataggccc tttgttgttc tctcggtact
     3421 tgttctgcag gaagatgtag gtggccaggg cccccatgga gtagaggaag gcaaacacag
     3481 ccacggtgac aaagaattca gccgacgagg agtagtcccc aaccaggaag atcttggtag
     3541 tgcccccttt gacgcaggag ggtgcatcaa agtacacttg gtgcagcctg aaggggtact
     3601 cgaattcaac ttcgatgttg agggcactct ccgtcttgtt ggcacactcc acgctcagcc
     3661 gaagctcccc ggtgtagctg ccacacgtag caaaggcgaa gatggcaaag acccactgca
     3721 gcaccttcac gaagccaagg ggctccttga ccacccggaa ctgaccccca gccaccagct
     3781 gattcaccac gtccatgcca ccggtggctc tagcataact tcgtataaag tatcctatac
     3841 gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaat
     3901 aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt atcgatgccc
     3961 gagataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     4021 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     4081 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttagttctt gccacggcgg
     4141 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     4201 attccgtggt gtttatttgt gaaatttgtg atgctattgc tttatttgta accatctagc
     4261 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     4321 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag
     4381 gttttttaaa gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     4441 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     4501 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     4561 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     4621 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4681 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4741 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4801 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     4861 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4921 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     4981 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5041 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     5101 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5161 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5221 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     5281 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     5341 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5401 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5461 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5521 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5581 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5641 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5701 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5761 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5821 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5881 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5941 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6001 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6061 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6121 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6181 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6241 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6301 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6361 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6421 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6481 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6541 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6601 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6661 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6721 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6781 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6841 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6901 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6961 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7021 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7081 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtcctg
     7141 caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc
     7201 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
     7261 catcactagg ggttcctgcg gccgcagatg ggggggatgc tgcac
//
