LOCUS       sequence-256581-        6961 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bicistronic plasmid for dual-color neuronal calcium and morphology 
            imaging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6961)
  TITLE     bicistronic plasmid for dual-color neuronal calcium and morphology 
            imaging
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6961)
  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            "Unpublished"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: pAAV-hSyn-DIO-jGCamp7b-P2A-GSG-mRuby3
FEATURES             Location/Qualifiers
     source          1..6961
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     polyA_signal    181..657
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     439..458
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     724..740
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     725..741
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    744..1332
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             850..861
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1259..1279
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(1345..1378)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1429..1462)
                     /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)."
     regulatory      1469..1478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1475..1483,1484..1501,1502..1504,1505..1537,1538..1540,
                     1541..1564,1565..1591,1592..1648,1649..1654,1655..1924,
                     1925..1942,1943..2377,2378..2383,2384..2824)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNPPDQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMYARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1571..1580
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1861..1882
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1988..2009)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2249..2268)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2834..2890
                     /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             2891..3601
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     protein_bind    3613..3646
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3697..3730
                     /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)."
     promoter        complement(3739..4186)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   4235..4364
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4426..5014)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4506..4525)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5185..5976,5977..6045))
                     /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     5808..5827
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6046..6150)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6218..6236
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6274..6296)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6396..6415
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6432..6887)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6569..6590)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6781..6800
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgct cggtccgcac gtggttacct acaaaatcag
      181 aaggacaggg aagggagcag tggttcacgc ctgtaatccc agcaatttgg gaggccaagg
      241 tgggtagatc acctgagatt aggagttgga gaccagcctg gccaatatgg tgaaaccccg
      301 tctctaccaa aaaaacaaaa attagctgag cctggtcatg catgcctgga atcccaacaa
      361 ctcgggaggc tgaggcagga gaatcgcttg aacccaggag gcggagattg cagtgagcca
      421 agattgtgcc actgcactcc agcttggttc ccaatagacc ccgcaggccc tacaggttgt
      481 cttcccaact tgccccttgc tccataccac ccccctccac cccataatat tatagaagga
      541 cacctagtca gacaaaatga tgcaacttaa ttttattagg acaaggctgg tgggcactgg
      601 agtggcaact tccagggcca ggagaggcac tggggagggg tcacagggat gccacccgta
      661 gatcatctct agactcgagg tcgaggccag agaggccatc gaatggcgcg ccgcgatcgc
      721 gtctcgaggt cgacggtatc gatgcgggga ggcggcccaa agggagatcc gactcgtctg
      781 agggcgaagg cgaagacgcg gaagaggccg cagagccggc agcaggccgc gggaaggaag
      841 gtccgctgga ttgagggccg aagggacgta gcagaaggac gtcccgcgca gaatccaggt
      901 ggcaacacag gcgagcagcc aaggaaagga cgatgatttc cccgacaaca ccacggaatt
      961 gtcagtgccc aacagccgag cccctgtcca gcagcgggca aggcaggcgg cgatgagttc
     1021 cgccgtggca atagggaggg ggaaagcgaa agtcccggaa aggagctgac aggtggtggc
     1081 aatgccccaa ccagtggggg ttgcgtcagc aaacacagtg cacaccacgc cacgttgcct
     1141 gacaacgggc cacaactcct cataaagaga cagcaaccag gatttataca aggaggagaa
     1201 aatgaaagcc atacgggaag caatagcatg atacaaaggc attaaagcag cgtatccaca
     1261 tagcgtaaaa ggagcaacat agttaagaat accagtcaat ctttcacaaa ttttgtaatc
     1321 cagaggttga ttatcgataa gcttataact tcgtatagca tacattatac gaagttattc
     1381 tttgcaccat tctaaagaat aacagtgata atttctgggt taaggcaaat aacttcgtat
     1441 aggatacttt atacgaagtt atcagctggc caccatgggt tctcatcatc atcatcatca
     1501 tggtatggct agcatgactg gtggacagca aatgggtcgg gatctgtacg acgatgacga
     1561 taaggatctc gccaccatgg tcgactcatc acgtcgtaag tggaataaga caggtcacgc
     1621 agtcagagct ataggtcggc tgagctcact cgagaacgtc tatatcaagg ccgacaagca
     1681 gaagaacggc atcaaggcga acttcaagat ccgccacaac atcgaggacg gcggcgtgca
     1741 gctcgcctac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1801 caaccactac ctgagcgtgc agtccaaact ttcgaaagac cccaacgaga agcgcgatca
     1861 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     1921 caagggcggt accggaggga gcatggtgag caagggcgag gagctgttca ccggggtggt
     1981 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2041 gggtgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     2101 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     2161 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2221 catccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2281 gaagttcgag ggcgacaccc tggtgaaccg catcgagctt aagggcatcg acttcaagga
     2341 ggacggcaac atcctggggc acaagctgga gtacaaccca cctgaccaac tgactgaaga
     2401 gcagatcgca gaatttaaag agcttttctc cctatttgac aaggacgggg atgggacaat
     2461 aacaaccaag gagctgggga cggtgatgcg gtctctgggg cagaacccca cagaagcaga
     2521 gctgcaggac atgatcaatg aagtagatgc cgacggtgac ggcacaatcg acttccctga
     2581 gttcctgaca atgtacgcaa gaaaaatgaa atacagggac acggaagaag aaattagaga
     2641 agcgttcggt gtgtttgata aggatggcaa tggctacatc agtgcagcag agcttcgcca
     2701 cgtgatgaca aaccttggag agaagttaac agatgaagag gttgatgaaa tgatcaggga
     2761 agcagacatc gatggggatg gtcaggtaaa ctacgaagag tttgtacaaa tgatgacagc
     2821 gaagggatcc ggagccacga acttctctct gttaaagcaa gcaggagacg tggaagaaaa
     2881 ccccggtcct atggtgtcta agggcgaaga gctgatcaag gaaaatatgc gtatgaaggt
     2941 ggtcatggaa ggttcggtca acggccacca attcaaatgc acaggtgaag gagaaggcag
     3001 accgtacgag ggaactcaaa ccatgaggat caaagtcatc gagggaggac ccctgccatt
     3061 tgcctttgac attcttgcca cgtcgttcat gtatggcagc cgtactttta tcaagtaccc
     3121 ggccgacatc cctgatttct ttaaacagtc ctttcctgag ggttttactt gggaaagagt
     3181 tacgagatac gaagatggtg gagtcgtcac cgtcacgcag gacaccagcc ttgaggatgg
     3241 cgagctcgtc tacaacgtca aggtcagagg ggtaaacttt ccctccaatg gtcccgtgat
     3301 gcagaagaag accaagggtt gggagcctaa tacagagatg atgtatccag cagatggtgg
     3361 tctgagagga tacactgaca tcgcactgaa agttgatggt ggtggccatc tgcactgcaa
     3421 cttcgtgaca acttacaggt caaaaaagac cgtcgggaac atcaagatgc ccggtgtcca
     3481 tgccgttgat caccgcctgg aaaggatcga ggagagtgac aatgaaacct acgtagtgca
     3541 acgcgaagtg gcagttgcca aatacagcaa ccttggtggt ggcatggacg agctgtacaa
     3601 gtgagtttaa acataacttc gtataatgta tgctatacga agttattaag aggtttcata
     3661 ttgctaatag cagctacaat ccagctacca ttctgcataa cttcgtataa agtatcctat
     3721 acgaagttat gaattcgact gcgctctcag gcacgacacg actcctccgc tgcccaccgc
     3781 agactgaggc agcgctgagt cgccggcgcc gcagcgcaga tggtcgcgcc cgtgcccccc
     3841 tatctcgcgc ctcgcgtggt gcggtccggc tgggccggcg gcggcgcgga cgcgaccaag
     3901 gtggccggga aggggagttt gcgggggacc ggcgagtgac gtcagcgcgc cttcagtgct
     3961 gaggcggcgg tggcgcgcgc cgccaggcgg gggcgaaggc actgtccgcg gtgctgaagc
     4021 tggcagtgcg cacgcgcctc gccgcatcct gtttcccctc cccctctctg ataggggatg
     4081 cgcaatttgg ggaatggggg ttgggtgctt gtccagtggg tcggggtcgg tcgtcaggta
     4141 ggcaccccca ccccgcctca tcctggtcct aaaacccact tgcactcata cgcagggccc
     4201 tctgcagctc gagtctagcg acgcgtgcgg ccgcaggaac ccctagtgat ggagttggcc
     4261 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     4321 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggacatgt gagcaaaagg
     4381 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4441 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4501 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4561 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4621 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4681 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4741 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4801 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4861 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4921 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4981 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5041 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5101 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5161 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5221 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5281 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5341 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5401 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5461 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5521 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5581 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5641 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5701 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5761 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5821 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5881 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5941 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6001 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6061 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6121 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6181 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     6241 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     6301 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     6361 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     6421 agtgcaccat aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     6481 aatcagctca ttttttaacc aatagaccga aatcggcaaa atcccttata aatcaaaaga
     6541 atagcccgag atagagttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     6601 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     6661 accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     6721 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     6781 agggaagaaa gcgaaaggag cgggcgctaa ggcgctggca agtgtagcgg tcacgctgcg
     6841 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtact atggttgctt
     6901 tgacgtatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     6961 c
//
