LOCUS Exported 7314 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION For cre-dependent enriched expression of GCaMP6s in axons with cytosolic expression of mRuby3. ACCESSION . VERSION . KEYWORDS pAAV-hSynapsin1-FLEx-axon-GCaMP6s-P2A-mRuby3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7314) AUTHORS Broussard GJ, Liang Y, Fridman M, Unger EK, Meng G, Xiao X, Ji N, Petreanu L, Tian L TITLE In vivo measurement of afferent activity with axon-specific calcium imaging. JOURNAL Nat Neurosci. 2018 Sep;21(9):1272-1280. doi: 10.1038/s41593-018-0211-4. Epub 2018 Aug 20. PUBMED 30127424 REFERENCE 2 (bases 1 to 7314) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7314 /organism="synthetic DNA construct" /mol_type="other DNA" intron 533..665 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" primer_bind 648..667 /label=pMT2-F /note="Synthetic intron, forward primer" protein_bind complement(678..711) /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)." protein_bind complement(774..807) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(830..1537) /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(1538..1594) /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(1604..2950) /codon_start=1 /product="improved fluorescent protein-based calcium sensor (Chen et al., 2013)" /label=GCaMP6s /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT GHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPV LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYETRAEVKFEGDTLVNRIEL KGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSL GQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGNG YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" primer_bind 2419..2440 /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2546..2567) /label=EGFP-C /note="EGFP, forward primer" regulatory 2848..2857 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 3046..3079 /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)." protein_bind 3150..3183 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 3225..3813 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3278..3298) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3696..3707) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3891..4012 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3902..3921) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3956..3975 /label=SV40pA-R /note="SV40 polyA, reverse primer" rep_origin 4481..4936 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4568..4587) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4778..4799 /label=F1ori-F /note="F1 origin, forward primer" promoter 4962..5066 /gene="bla" /label=AmpR promoter CDS 5067..5927 /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(5285..5304) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6098..6686 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6587..6606 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6840..6857 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6973..6994 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 7009..7039 /label=lac promoter /note="promoter for the E. coli lac operon" repeat_region 7125..7254 /label=AAV2 ITR ORIGIN 1 gagctccagt gtgctggaat tcgccctttt cagtatttaa attgacgacc gaccccgacc 61 cactggacaa gcacccaacc cccattcccc aaattgcgca tcccctatca gagaggggga 121 ggggaaacag gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc 181 cttcgccccc gcctggcggc gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt 241 cactcgccgg tcccccgcaa actccccttc ccggccacct tggtcgcgtc cgcgccgccg 301 ccggcccagc cggaccgcac cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc 361 tgcgctgcgg cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg 421 tgtcgtgcct gagagcgcag ctgtgctcct gggcaccgcg cagtccgccc ccgcggctcc 481 tggccagacc acccctagga ccccctgccc caagtcgcag ccgttggatc aggtaagtat 541 caaggttaca agacaggttt aaggagacca atagaaactg ggcttgtcga gacagagaag 601 actcttgcgt ttctgatagg cacctattgg tcttactgac atccactttg cctttctctc 661 cacaggtgat gatccccata acttcgtata aagtatccta tacgaagtta tatcaaaata 721 ggaagaccaa tgcttcacca tcgacccgaa ttgccaagca tcaccatcga cacataactt 781 cgtataatgt atgctatacg aagttatact aggagctcgg cgcgccttac ttgtacagct 841 cgtccatgcc accaccaagg ttgctgtatt tggcaactgc cacttctctt tgcactacgt 901 aggtttcatt gtcactctcc tcgatccttt ccaggcggtg atcaacggca tggacaccgg 961 gcatcttgat gttcccgacg gtcttttttg acctgtaagt tgtcacgaag ttgcagtgca 1021 gatggccacc accatcaact ttcagtgcga tgtcagtgta tcctctcaga ccaccatctg 1081 ctggatacat catctctgta ttaggctccc aacccttggt cttcttctgc atcacgggac 1141 cattggaggg aaagtttacc cctctgacct tgacgttgta gacgagctcg ccatcctcaa 1201 ggctggtgtc ctgcgtgacg gtgacgactc caccatcttc gtatctcgta actctttccc 1261 aagtaaaacc ctcaggaaag gactgtttaa agaaatcagg gatgtcggcc gggtacttga 1321 taaaggtacg gctgccatac atgaacgacg tggcaagaat gtcaaaggca aatggcaggg 1381 gtcctccctc gatgactttg atcctcatgg tttgcactcc ctcgtacggt ctgccttctc 1441 cttcacctgt gcatttgaat tggtggccgt tgaccgaacc ttccatgacc accttcatac 1501 gcatattttc cttgatcagc tcttcgccct tagacacagg accggggttt tcttccacgt 1561 ctcctgcttg ctttaacaga gagaagttcg tggctccgga tcccttcgct gtcatcattt 1621 gtacaaactc ttcgtagttt acctgaccat ccccatcgat gtctgcttcc ctgatcattt 1681 catcaacctc ttcatctgtt aacttctctc caaggtttgt catcacgtgg cgaagctctg 1741 ctgcactgat gtagccattg ccatccttat caaacacacc gaacgcttct ctaatttctt 1801 cttccgtgtc cctgtatttc atttttcttg ccatcattgt caggaactca gggaagtcga 1861 ttgtgccgtc accgtcggca tctacttcat tgatcatgtc ctgcagctct gcttctgtgg 1921 ggttctgccc cagagaccgc atcaccgtcc ccagctcctt ggttgttatt gtcccatccc 1981 cgtccttgtc aaatagggag aaagcctctt taaattctgc gatctgctct tcagtcagtt 2041 ggtccggcag gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga 2101 tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg 2161 tctcgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggatgtag ccttcgggca 2221 tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca 2281 cgccgtaggt cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc 2341 agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcaccctcg ccggacacgc 2401 tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga 2461 acagctcctc gcccttgctc accatgctcc ctccggtacc gcccttgtac agctcgtcca 2521 tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct 2581 cgttggggtc tttcgaaagt ttggactgca cgctcaggta gtggttgtcg ggcagcagca 2641 cggggccgtc gccgatgggg gtgttctgct ggtagtggta ggcgagctgc acgccgccgt 2701 cctcgatgtt gtggcggatg tggaagttcg ccttgatgcc gttcttctgc ttgtcggcct 2761 tgatatagac gttctcgagt gagctcagcc gacctatagc tctgactgcg tgacctgtct 2821 tattccactt acgacgtgat gagtcgacca tggtggcgag atccttatcg tcatcgtcgt 2881 acagatcccg acccatttgc tgtccaccag tcatgctagc cataccatga tgatgatgat 2941 gatgagaacc aatcttttgg tcctcatcat tcttttcaac ctgtttggtt cttctcatac 3001 agcacagcat ggtggcgact agtgctagcg gtaccgaatt ctagtataac ttcgtatagg 3061 atactttata cgaagttatc attgggattc ttcctatttt gatccaagca tcaccatcga 3121 ccctctagtc cagatctcac catcgaccca taacttcgta tagcatacat tatacgaagt 3181 tatgtccctc gaagaggttc gaattcgata tcaagcttat cgataatcaa cctctggatt 3241 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 3301 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 3361 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 3421 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 3481 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 3541 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 3601 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 3661 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 3721 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 3781 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgaccc gggcggccgc 3841 tcgagcatgc atctagagtc gacccgggcg gccgcttcga gcagacatga taagatacat 3901 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 3961 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa 4021 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa 4081 gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt cctagagcat ggctacgtag 4141 ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca 4201 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 4261 cgggctttgc ccgggcggcc tcagtgagcg agcgagggag tggccactcc ctctctgcgc 4321 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg 4381 gcggcctcag tgagcgagcg agcgcgcagc tggcgtaata gcgaagaggc ccgcaccgat 4441 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca 4501 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 4561 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 4621 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 4681 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 4741 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 4801 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 4861 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 4921 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 4981 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 5041 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 5101 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 5161 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 5221 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 5281 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 5341 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 5401 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 5461 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 5521 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 5581 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 5641 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 5701 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 5761 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 5821 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 5881 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 5941 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 6001 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 6061 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 6121 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 6181 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 6241 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 6301 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 6361 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 6421 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 6481 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 6541 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 6601 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 6661 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 6721 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 6781 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 6841 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc 6901 gttggccgat tcattaatgc agctgcgcgc tcgctcgctc aactgggccg cgcgcggtga 6961 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat 7021 gcttccggct cgtatgttgt gtggaattgt gagcggatga cagtgtaaga ccgccaacag 7081 ctaaraccgc ctctccccgc gcgttggccg attcattaat gcagctgcgc gctcgctcgc 7141 tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag 7201 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt 7261 taatgattaa cccgccatgc tacttatcta cgtagccatg ctctaggaag atcc //