LOCUS Exported 8747 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION QUAS reporter line that expresses cytosolic dTomato and the GCaMP6s calcium indicator, separated by T2A. ACCESSION . VERSION . KEYWORDS 15x-QUAS-dTomato-T2A-GCaMP6s SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8747) AUTHORS Matthews BJ, Younger MA, Vosshall LB TITLE The ion channel ppk301 controls freshwater egg-laying in the mosquito Aedes aegypti. JOURNAL Elife. 2019 May 21;8. pii: 43963. doi: 10.7554/eLife.43963. PUBMED 31112133 REFERENCE 2 (bases 1 to 8747) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8747 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 104..166 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" regulatory 652..661 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 658..1377 /codon_start=1 /product="enhanced CFP" /label=ECFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" polyA_signal 1500..1621 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter 2033..2271 /label=hsp70 promoter /note="Drosophila melanogaster hsp70Bb promoter" primer_bind 2218..2239 /label=pCasper-hs /note="Drosophila Hsp70 promoter, forward primer" CDS 2347..3048 /codon_start=1 /product="dimeric variant of DsRed fluorescent protein (Shaner et al., 2004)" /label=dTomato /note="mammalian codon-optimized" /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH QALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH HLFLYGMDELYK" primer_bind complement(2480..2498) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2748..2767 /label=mCherry-F /note="mCherry, forward primer" primer_bind 2942..2965 /label=DsRed1-C /note="DsRed1, forward primer" CDS 3052..3105 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 3106..4458 /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 3202..3211 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" intron 4535..4600 /label=small t intron /note="SV40 (simian virus 40) small t antigen intron" CDS 4730..4750 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 5022..5156 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" repeat_region 5431..5465 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(5956..5972) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5956..5972) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5969..5991) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5980..5996 /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)." promoter complement(6004..6034) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6049..6070 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6187..6204) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6358..6946) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6438..6457) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7117..7977) /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 7740..7759 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7978..8082) /gene="bla" /label=AmpR promoter rep_origin complement(8108..8563) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8245..8266) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 8457..8476 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8690..8712 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 8704..8721 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 8705..8721 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 tgaacccgtg gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca 61 gatgaagatg ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca 121 tattgtgacg tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct 181 gtatatcgag gtttatttat taatttgaat agatattaag ttttattata tttacactta 241 catactaata ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac 301 aaaaactcaa aatttcttct ataaagtaac aaaactttta tcgaattcct gcagcccggg 361 ggatccacta gttctagtgt tcccacaatg gttaattcga gctcgcccgg ggatctaatt 421 caattagaga ctaattcaat tagagctaat tcaattagga tccaagctta tcgatttcga 481 accctcgacc gccggagtat aaatagaggc gcttcgtcta cggagcgaca attcaattca 541 aacaagcaaa gtgaacacgt cgctaagcga aagctaagca aataaacaag cgcagctgaa 601 caagctaaac aatcggggta ccgctagagt cgacggtacg atccaccggt cgccaccatg 661 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 721 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 781 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 841 gtgaccaccc tgacctgggg cgtgcagtgc ttcagccgct accccgacca catgaagcag 901 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 961 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 1021 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 1081 ctggagtaca actacatcag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc 1141 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 1201 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 1261 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 1321 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc 1381 ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta cttgctttaa 1441 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta 1501 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 1561 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 1621 aaagcttatc gatacgcgta cggcactaga gcggccgcca ccgcggtcac gtgtcactgg 1681 gtcagtgggt aatcgcttat cctcggataa acaattatcc tcacgggtaa tcgcttatcc 1741 gctcgggtaa tcgcttatcc tcgggtaatc gcttatcctt gggtaatcgc ttatcctcgg 1801 ataaacaatt atcctcacgg gtaatcgctt atccgctcgg gtaatcgctt atcctcgggt 1861 aatcgcttat ccttgggtaa tcgcttatcc tcggataaac aattatcctc acgggtaatc 1921 gcttatccgc tcgggtaatc gcttatcctc gggtaatcgc ttatcctttc acgttgggac 1981 tcagtgagga ggacctgaat tcctgcagcc cgagcggaga ctctagcgag cgccggagta 2041 taaatagagg cgcttcgtct acggagcgac aattcaattc aaacaagcaa agtgaacacg 2101 tcgctaagcg aaagctaagc aaataaacaa gcgcagctga acaagctaaa caatctgcag 2161 taaagtgcaa gttaaagtga atcaattaaa agtaaccagc aaccaagtaa atcaactgca 2221 actactgaaa tctgccaaga agtaattatt gaatacaaga agagaactct gaatagggaa 2281 ttgggaattc gttaacagat cgggggatca attcgttaac agatctgcgg ccgcggctcg 2341 agcaaaatgg tgagcaaggg cgaggaggtc atcaaagagt tcatgcgctt caaggtgcgc 2401 atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 2461 tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc 2521 tgggacatcc tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc 2581 gacatccccg attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 2641 aacttcgagg acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg 2701 ctgatctaca aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag 2761 aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg 2821 aagggcgaga tccaccaggc cctgaagctg aaggacggcg gccgctacct ggtggagttc 2881 aagaccatct acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc 2941 aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc 3001 gagggccgcc accacctgtt cctgtacggc atggacgagc tgtacaaggg agagggcaga 3061 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gccctatggg ttctcatcat 3121 catcatcatc atggtatggc tagcatgact ggtggacagc aaatgggtcg ggatctgtac 3181 gacgatgacg ataaggatct cgccaccatg gtcgactcat cacgtcgtaa gtggaataag 3241 acaggtcacg cagtcagagc tataggtcgg ctgagctcac tcgagaacgt ctatatcaag 3301 gccgacaagc agaagaacgg catcaaggcg aacttccaca tccgccacaa catcgaggac 3361 ggcggcgtgc agctcgccta ccactaccag cagaacaccc ccatcggcga cggccccgtg 3421 ctgctgcccg acaaccacta cctgagcgtg cagtccaaac tttcgaaaga ccccaacgag 3481 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 3541 gacgagctgt acaagggcgg taccggaggg agcatggtga gcaagggcga ggagctgttc 3601 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 3661 gtgtccggcg agggtgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 3721 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg 3781 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 3841 cccgaaggct acatccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 3901 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 3961 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacct gccggaccaa 4021 ctgactgaag agcagatcgc agaatttaaa gaggctttct ccctatttga caaggacggg 4081 gatgggacaa taacaaccaa ggagctgggg acggtgatgc ggtctctggg gcagaacccc 4141 acagaagcag agctgcagga catgatcaat gaagtagatg ccgacggtga cggcacaatc 4201 gacttccctg agttcctgac aatgatggca agaaaaatga aatacaggga cacggaagaa 4261 gaaattagag aagcgttcgg tgtgtttgat aaggatggca atggctacat cagtgcagca 4321 gagcttcgcc acgtgatgac aaaccttgga gagaagttaa cagatgaaga ggttgatgaa 4381 atgatcaggg aagcagacat cgatggggat ggtcaggtaa actacgaaga gtttgtacaa 4441 atgatgacag cgaagtgaga tctttgtgaa ggaaccttac ttctgtggtg tgacataatt 4501 ggacaaacta cctacagaga tttaaagctc taaggtaaat ataaaatttt taagtgtata 4561 atgtgttaaa ctactgattc taattgtttg tgtattttag attccaacct atggaactga 4621 tgaatgggag cagtggtgga atgcctttaa tgaggaaaac ctgttttgct cagaagaaat 4681 gccatctagt gatgatgagg ctactgctga ctctcaacat tctactcctc caaaaaagaa 4741 gagaaaggta gaagacccca aggactttcc ttcagaattg ctaagttttt tgagtcatgc 4801 tgtgtttagt aatagaactc ttgcttgctt tgctatttac accacaaagg aaaaagctgc 4861 actgctatac aagaaaatta tggaaaaata tttgatgtat agtgccttga ctagagatca 4921 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc 4981 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt 5041 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 5101 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcgata 5161 tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa 5221 tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt 5281 gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct 5341 cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat 5401 ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc 5461 tagggttaat ctagctgcat caggatcata tcgtcgggtc ttttttccgg ctcagtcatc 5521 gcccaagctg gcgctatctg ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg 5581 ttgaagcggc atggaaagag tttgccgagg atgactgctg ctgcattgac gttgagcgaa 5641 aacgcacgtt taccatgatg attcgggaag gtgtggccat gcacgccttt aacggtgaac 5701 tgttcgttca ggccacctgg gataccagtt cgtcgcggct tttccggaca cagttccgga 5761 tggtcagccc gaagcgcatc agcaacccga acaataccgg cgacagccgg aactgccgtg 5821 ccggtgtgca gattaatgac agcggtgcgg cgctgggata ttacgtcagc gaggacgggt 5881 atcctggctg gatgccgcag aaatggacat ggataccccg tgagttaccc ggcgggcgcg 5941 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 6001 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 6061 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 6121 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 6181 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 6241 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 6301 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 6361 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 6421 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 6481 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 6541 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 6601 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 6661 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 6721 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 6781 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 6841 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 6901 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 6961 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 7021 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 7081 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 7141 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 7201 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 7261 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 7321 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 7381 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 7441 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 7501 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 7561 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 7621 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 7681 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 7741 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 7801 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 7861 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 7921 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 7981 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 8041 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 8101 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc 8161 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag 8221 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg 8281 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca 8341 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa 8401 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg 8461 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta 8521 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg 8581 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg 8641 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga 8701 cgttgtaaaa cgacggccag tgagcgcgcc tcgttcattc acgtttt //