LOCUS Exported 6961 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION bicistronic plasmid for dual-color neuronal calcium and morphology imaging. ACCESSION . VERSION . KEYWORDS pAAV-hSyn-DIO-jGCamp7b-P2A-GSG-mRuby3 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REFERENCE 1 (bases 1 to 6961) TITLE bicistronic plasmid for dual-color neuronal calcium and morphology imaging JOURNAL Unpublished REFERENCE 2 (bases 1 to 6961) AUTHORS .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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6961 /organism="synthetic DNA construct" /mol_type="other DNA" 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 1345..1378 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 1429..1462 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." regulatory 1469..1478 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1475..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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" 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 complement(3613..3646) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(3697..3730) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). 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(5185..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 //