LOCUS       sequence-200277-        6265 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Bicistronic vector expressing mRuby2 and GCaMP6f from a single open 
            reading frame. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6265)
  AUTHORS   Rose T, Jaepel J, Hubener M, Bonhoeffer T
  TITLE     Cell-specific restoration of stimulus preference after monocular 
            deprivation in the visual cortex.
  JOURNAL   Science. 2016 Jun 10;352(6291):1319-22. doi: 
            10.1126/science.aad3358.
  PUBMED    27284193
REFERENCE   2  (bases 1 to 6265)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2016-06-10- 10"; volume: "352"; 
            issue: "6291"; pages: "1319-22"
COMMENT     Sequence Label: pAAV-hSyn1-mRuby2-GSG-P2A-GCaMP6f-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..6265
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        211..658
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      694..703
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             700..1410
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             1420..1476
                     /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             join(1477..1482,1483..1500,1501..1503,1504..1536,1537..1539,
                     1540..1563,1564..1590,1591..1647,1648..1653,1654..1923,
                     1924..1941,1942..2376,2377..2382,2383..2826)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT
                     GHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRSL
                     GQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGNG
                     YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1570..1579
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1860..1881
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1987..2008)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2248..2267)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2851..3439
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2904..2924)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3322..3333)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3442..3458)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3443..3459)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3488..3609
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3499..3518)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3553..3572
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3988..4576)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4068..4087)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4747..5538,5539..5607))
                     /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     5370..5389
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5608..5712)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5738..6193)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5875..5896)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6087..6106
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg
       61 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
      121 ggggttcctt gtagttaatg attaacccgc catgctactt atctacgtag ccatgctcta
      181 ggaagatctc tgcagagggc cctgcgtatg agtgcaagtg ggttttagga ccaggatgag
      241 gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc acccaacccc
      301 cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga tgcggcgagg
      361 cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc
      421 gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac
      481 tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca
      541 cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc
      601 agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga gagcgcagtc
      661 gaattcaagc tgctagctag taccggtcct agggccacca tggtgtctaa gggcgaagag
      721 ctgatcaagg aaaatatgcg tatgaaggtg gtcatggaag gttcggtcaa cggccaccaa
      781 ttcaaatgca caggtgaagg agaaggcaat ccgtacatgg gaactcaaac catgaggatc
      841 aaagtcatcg agggaggacc cctgccattt gcctttgaca ttcttgccac gtcgttcatg
      901 tatggcagcc gtacttttat caagtacccg aaaggcattc ctgatttctt taaacagtcc
      961 tttcctgagg gttttacttg ggaaagagtt acgagatacg aagatggtgg agtcgtcacc
     1021 gtcatgcagg acaccagcct tgaggatggc tgtctcgttt accacgtcca agtcagaggg
     1081 gtaaactttc cctccaatgg tcccgtgatg cagaagaaga ccaagggttg ggagcctaat
     1141 acagagatga tgtatccagc agatggtggt ctgaggggat acactcatat ggcactgaaa
     1201 gttgatggtg gtggccatct gtcttgctct ttcgtaacaa cttacaggtc aaaaaagacc
     1261 gtcgggaaca tcaagatgcc cggtatccat gccgttgatc accgcctgga aaggttagag
     1321 gaaagtgaca atgaaatgtt cgtagtacaa cgcgaacacg cagttgccaa gttcgccggg
     1381 cttggtggtg ggatggacga gctgtacaag ggatccggag ccacgaactt ctctctgtta
     1441 aagcaagcag gagacgtgga agaaaacccc ggtcctggtt ctcatcatca tcatcatcat
     1501 ggtatggcta gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat
     1561 aaggatctcg ccaccatggt cgactcatca cgtcgtaagt ggaataagac aggtcacgca
     1621 gtcagagcta taggtcggct gagctcactc gagaacgtct atatcaaggc cgacaagcag
     1681 aagaacggca tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag
     1741 ctcgcctacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1801 aaccactacc tgagcgtgca gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac
     1861 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1921 aagggcggta ccggagggag catggtgagc aagggcgagg agctgttcac cggggtggtg
     1981 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     2041 ggtgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     2101 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     2161 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     2221 atccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     2281 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2341 gacggcaaca tcctggggca caagctggag tacaacctgc cggaccaact gactgaagag
     2401 cagatcgcag aatttaaaga ggaattctcc ctatttgaca aggacgggga tgggacaata
     2461 acaaccaagg agctggggac ggtgatgcgg tctctggggc agaaccccac agaagcagag
     2521 ctgcaggaca tgatcaatga agtagatgcc gacggtgacg gcacaatcga cttccctgag
     2581 ttcctgacaa tgatggcaag aaaaatgaaa tacagggaca cggaagaaga aattagagaa
     2641 gcgttcggtg tgtttgataa ggatggcaat ggctacatca gtgcagcaga gcttcgccac
     2701 gtgatgacaa accttggaga gaagttaaca gatgaagagg ttgatgaaat gatcagggaa
     2761 gcagacatcg atggggatgg tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg
     2821 aagtaagcgg ccgcactaga gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     2881 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     2941 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3001 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3061 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3121 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3181 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3241 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     3301 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3361 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     3421 ctttgggccg cctccccgca tcgataccgt cgacctcgac ccgggcggcc gcttcgagca
     3481 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     3541 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     3601 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg
     3661 gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatcttcct
     3721 agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct
     3781 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgact cgctgcgctc
     3841 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3901 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3961 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4021 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4081 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4141 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4201 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4261 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4321 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4381 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4441 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4501 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4561 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4621 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4681 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4741 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4801 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4861 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4921 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4981 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5041 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5101 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5161 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5221 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5281 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5341 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5401 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5461 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5521 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5581 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5641 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5701 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     5761 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     5821 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     5881 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     5941 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     6001 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     6061 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     6121 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     6181 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     6241 cgggcctctt cgctattacg ccagc
//
