LOCUS Exported 6730 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION For co-expression of GCaMP6s with mRuby3. ACCESSION . VERSION . KEYWORDS pAAV-hSynapsin1-GCaMP6s-P2A-mRuby3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6730) 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 6730) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6730 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 26..155 /label=AAV2 ITR promoter 237..684 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" regulatory 715..724 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 721..2070 /codon_start=1 /product="improved fluorescent protein-based calcium sensor (Chen et al., 2013)" /label=GCaMP6s /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" regulatory 817..826 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 1107..1128 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1234..1255) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1495..1514) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" CDS 2080..2136 /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 2137..2844 /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" misc_feature 2868..3456 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2921..2941) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3339..3350) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3459..3475) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3460..3476) /label=pBluescriptKS /note="For pBluescript vector" polyA_signal 3505..3626 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3516..3535) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3570..3589 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 3788..3917 /label=AAV2 ITR primer_bind complement(3936..3953) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3936..3952) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3945..3967) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4094..4549 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4181..4200) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4391..4412 /label=F1ori-F /note="F1 origin, forward primer" promoter 4575..4679 /gene="bla" /label=AmpR promoter CDS 4680..5540 /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(4898..4917) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5711..6299 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6200..6219 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6453..6470 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6587..6608 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6623..6653 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6661..6677 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 6666..6688 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6685..6701 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6685..6701 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 cgcagaattt aaagaggtaa ttaggctgcg cgctcgctcg ctcactgagg ccgcccgggc 61 aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag 121 agagggagtg gccaactcca tcactagggg ttccttgtag ttaatgatta acccgccatg 181 ctacttatct acgtagccat gctctaggaa gatctctgca gagggccctg cgtatgagtg 241 caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac gaccgacccc 301 gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg 361 gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca 421 gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg 481 acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc 541 gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac 601 catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggagga 661 gtcgtgtcgt gcctgagagc gcagtcgaat tcaagctgct agctagtacc ggtcgccacc 721 atgggttctc atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg 781 ggtcgggatc tgtacgacga tgacgataag gatctcgcca ccatggtcga ctcatcacgt 841 cgtaagtgga ataagacagg tcacgcagtc agagctatag gtcggctgag ctcactcgag 901 aacgtctata tcaaggccga caagcagaag aacggcatca aggcgaactt ccacatccgc 961 cacaacatcg aggacggcgg cgtgcagctc gcctaccact accagcagaa cacccccatc 1021 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcgtgcagtc caaactttcg 1081 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 1141 atcactctcg gcatggacga gctgtacaag ggcggtaccg gagggagcat ggtgagcaag 1201 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 1261 ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg caagctgacc 1321 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 1381 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 1441 ttcaagtccg ccatgcccga aggctacatc caggagcgca ccatcttctt caaggacgac 1501 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 1561 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 1621 aacctgccgg accaactgac tgaagagcag atcgcagaat ttaaagaggc tttctcccta 1681 tttgacaagg acggggatgg gacaataaca accaaggagc tggggacggt gatgcggtct 1741 ctggggcaga accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac 1801 ggtgacggca caatcgactt ccctgagttc ctgacaatga tggcaagaaa aatgaaatac 1861 agggacacgg aagaagaaat tagagaagcg ttcggtgtgt ttgataagga tggcaatggc 1921 tacatcagtg cagcagagct tcgccacgtg atgacaaacc ttggagagaa gttaacagat 1981 gaagaggttg atgaaatgat cagggaagca gacatcgatg gggatggtca ggtaaactac 2041 gaagagtttg tacaaatgat gacagcgaag ggatccggag ccacgaactt ctctctgtta 2101 aagcaagcag gagacgtgga agaaaacccc ggtcctgtgt ctaagggcga agagctgatc 2161 aaggaaaata tgcgtatgaa ggtggtcatg gaaggttcgg tcaacggcca ccaattcaaa 2221 tgcacaggtg aaggagaagg cagaccgtac gagggagtgc aaaccatgag gatcaaagtc 2281 atcgagggag gacccctgcc atttgccttt gacattcttg ccacgtcgtt catgtatggc 2341 agccgtacct ttatcaagta cccggccgac atccctgatt tctttaaaca gtcctttcct 2401 gagggtttta cttgggaaag agttacgaga tacgaagatg gtggagtcgt caccgtcacg 2461 caggacacca gccttgagga tggcgagctc gtctacaacg tcaaggtcag aggggtaaac 2521 tttccctcca atggtcccgt gatgcagaag aagaccaagg gttgggagcc taatacagag 2581 atgatgtatc cagcagatgg tggtctgaga ggatacactg acatcgcact gaaagttgat 2641 ggtggtggcc atctgcactg caacttcgtg acaacttaca ggtcaaaaaa gaccgtcggg 2701 aacatcaaga tgcccggtgt ccatgccgtt gatcaccgcc tggaaaggat cgaggagagt 2761 gacaatgaaa cctacgtagt gcaaagagaa gtggcagttg ccaaatacag caaccttggt 2821 ggtggcatgg acgagctgta caagtaagct tactagagct tatcgataat caacctctgg 2881 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 2941 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 3001 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 3061 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg 3121 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 3181 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 3241 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca 3301 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 3361 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 3421 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgacccg 3481 ggcggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta 3541 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 3601 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 3661 ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa 3721 tcgataagga tcttcctaga gcatggctac gtagataagt agcatggcgg gttaatcatt 3781 aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 3841 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg 3901 agcgagcgag cgcgcagcct taattaacct aattcactgg ccgtcgtttt acaacgtcgt 3961 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 4021 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 4081 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 4141 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 4201 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 4261 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 4321 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 4381 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 4441 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 4501 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt 4561 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 4621 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 4681 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 4741 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 4801 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 4861 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 4921 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 4981 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 5041 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 5101 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 5161 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 5221 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 5281 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 5341 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 5401 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 5461 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 5521 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 5581 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 5641 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 5701 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 5761 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 5821 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 5881 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 5941 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 6001 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 6061 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 6121 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 6181 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 6241 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 6301 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 6361 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 6421 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 6481 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 6541 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 6601 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 6661 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta 6721 attaaggcct //