LOCUS Exported 6265 bp ds-DNA linear SYN 25-MAY-2021 DEFINITION Bicistronic vector expressing mRuby2 and GCaMP6f from a single open reading frame. . ACCESSION . VERSION . KEYWORDS pAAV-hSyn1-mRuby2-GSG-P2A-GCaMP6f-WPRE-pA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct 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 May 25, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6265 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 211..658 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" regulatory 694..703 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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 1477..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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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" primer_bind complement(3817..3834) /label=L4440 /note="L4440 vector, forward 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(4747..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 //