LOCUS Exported 8495 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression of scFLARE1 component bearing a p2A-eGFP as transfection marker. ACCESSION . VERSION . KEYWORDS scFLARE1-p2A-eGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8495) AUTHORS Sanchez MI, Nguyen QA, Wang W, Soltesz I, Ting AY TITLE Transcriptional readout of neuronal activity via an engineered Ca(2+)-activated protease. JOURNAL Proc Natl Acad Sci U S A. 2020 Dec 15. pii: 2006521117. doi: 10.1073/pnas.2006521117. PUBMED 33323488 REFERENCE 2 (bases 1 to 8495) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8495 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..304 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 305..508 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 459..479 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 553..572 /label=T7 /note="T7 promoter, forward primer" promoter 553..571 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 585..594 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" sig_peptide 591..653 /product="leader sequence from mouse immunoglobulin kappa light chain" /label=Ig-kappa leader CDS 3645..3668 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 3669..4106 /codon_start=1 /gene="Saccharomyces cerevisiae GAL4 (truncated)" /product="DNA binding domain of the GAL4 transcriptional activator" /label=GAL4 DNA binding domain /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS" CDS 4107..4448 /codon_start=1 /gene="Saccharomyces cerevisiae GAL4 (truncated)" /product="activation domain of the GAL4 transcriptional activator" /label=GAL4 activation domain /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD EDTPPNPKKE" CDS 4449..4490 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 4500..4556 /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 4563..5279 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4608..4629) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4869..4888) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5216..5237 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(5316..5333) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5322..5529 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5558..5698 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5558..5687 /label=AAV2 ITR rep_origin 5773..6228 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5860..5879) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6070..6091 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6245..6264) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6364..6386 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6424..6442) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6510..6614 /gene="bla" /label=AmpR promoter CDS 6615..7475 /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(6833..6852) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7646..8234 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8135..8154 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 8296..8425 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 481 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg 541 gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgccacc atggagacag 601 acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt gacggtgccc 661 agccggccct cgacttccag aaggcctcca gcatagtcta taagaaagag ggggaacagg 721 tggagttctc cttcccactc gcctttacag ttgaaaagct gacgggcagt ggcgagctgt 781 ggtggcaggc ggagagggct tcctcctcca agtcttggat cacctttgac ctgaagaaca 841 aggaagtgtc tgtaaaacgg gttacccagg accctaagct ccagatgggc aagaagctcc 901 cgctccacct caccctgccc caggccttgc ctcagtatgc tggctctgga aacctcaccc 961 tggcccttga agcgaaaaca ggaaagttgc atcaggaagt gaacctggtg gtgatgagag 1021 ccactcagct ccagaaaaat ttgacctgtg aggtgtgggg acccacctcc cctaagctga 1081 tgctgagctt gaaactggag aacaaggagg caaaggtctc gaagcgggag aaggcggtgt 1141 gggtgctgaa ccctgaggcg gggatgtggc agtgtctgct gagtgactcg ggacaggtcc 1201 tgctggaatc caacatcaag gttctgccca catggtccac cccggtgcag ccaatggccc 1261 tgattgtgct ggggggcgtc gccggcctcc tgcttttcat tgggctaggc atcttcttct 1321 gtgtcaggtg ccggcaccga aggcgcaagg gctcgggctc gacctcgggc tcgggcagcg 1381 gtggctctag aggttctggt ggcagctctg gaggtatgaa tggagctata ggaggtgacc 1441 ttttgctcaa ttttcctgac atgtcggtcc tagagcgcca aagggctcac ctcaagtacc 1501 tcaatcccac ctttgattct cctctcgccg gcttctttgc cgattcttca atgattaccg 1561 gcggcgagat ggacagctat ctttcgactg ccggtttgaa tcttccgatg atgtacggtg 1621 agacgacggt ggaaggtgat tcaagactct caatttcgcc ggaaacgacg cttgggactg 1681 gaaatttcaa ggcagcgaag tttgatacag agactaagga ttgtaatgag gcggcgaaga 1741 agatgacgat gaacagagat gacctagtag aagaaggaga agaagagaag tcgaaaataa 1801 cagagcaaaa caatgggagc acaaaaagca tcaagaagat gaaacacaaa gccaagaaag 1861 aagagaacaa tttctctaat gattcatcta aagtgacgaa ggaattggag aaaacggatt 1921 atattcatgg tggctctggt agcggagaaa gcttgtttaa gggaccacgt gattacaacc 1981 cgatatcgag caccatttgt catttgacga atgaatctga tgggcacaca acatcgttgt 2041 atggtattgg atttggtccc ttcatcatta caaacaagca cttgtttaga agaaataatg 2101 gaacactgtt ggtccaatca ctacatctcg aggaccaact gactgaagag cagatcgcag 2161 aatttaaaga ggctttctcc ctatttgaca aggacgggga tgggacaata acaaccaagg 2221 agctggggac ggtgatgcgg tctctggggc agaaccccac agaagcagag ctgcaggaca 2281 tgatcaatga agtagatgcc gacggtgacg gcacaatcga cttccctgag ttcctgacaa 2341 tgatggcaag aaaaatgaaa tacacaggcg gtgtgaagct gatcccctcc tggaccaccg 2401 tgatcctggt gaagagcatg ctgcgcaagc gcagcttcgg caaccccttc ggtggcgaca 2461 gtgaagaaga aattagagaa gcgttccgtg tgtttgataa ggatggcaat ggctacatca 2521 gtgcagcaga gcttcgccac gtgatgacaa accttggaga gaagttaaca gatgaagagg 2581 ttgatgaaat gatcagggaa gcagacatcg atggggatgg tcaggtaaac tacgaagagt 2641 ttgtacaaat gatgacagcg aaggcggccg cgggtgtatt caaggtcaag aacaccacga 2701 ctttgcaaca acacctcatt gatgggaggg acatgataat tattcgcatg cctaaggatt 2761 tcccaccatt tcctcaaaag ctgaaattta gagagccaca aagggaagag cgcatatgtc 2821 ttgtgacaac caacttccaa actaagagca tgtctagcat ggtgtcagac actagttgca 2881 cattcccttc atctgatggc atattctgga agcattggat tcaaaccaag gatgggcagt 2941 gtggcaatcc attagtatca actagagatg ggttcattgt tggtatacac tcagcatcga 3001 atttcaccaa cacaaacaat tatttcacaa gcgtgccgaa aaacttcatg gaattgttga 3061 caaatcagga ggcgcagcag tgggttagtg gttggcgatt aaatgctgac tcagtattgt 3121 ggggtggcca taaagttttc atggtggagc tggccgagaa gctggccggc ctggacatca 3181 acggtggagc tagcgagttt cgggcaacca cactggaacg gatcgagaaa agcttcgtga 3241 ttactgatcc gagactgcct gacaacccaa tcatttttgt gagcgattcc ttcctgcagc 3301 tgacagaata ttctcgggaa gagatcctgg ggcgcaattg ccgttttctg cagggacccg 3361 agacagaccg tgccactgtt cggaaaatca gagatgctat tgacaaccag actgaagtga 3421 ccgttcagct gatcaattat accaagagcg gcaagaagtt ctggaacgtg ttccacctgc 3481 agccgatgcg cgattataag ggcgacgtcc agtacttcat tggcgtgcag ctggatggca 3541 ccgaacgtct tcatggcgcc gctgagcgtg aggcggtctg cctggtcaaa aagacagcct 3601 ttcagattgc tgagaacctg tacttccaga tgggcggagg atccgactac aaggatgacg 3661 acgataagaa gctactgtct tctatcgaac aagcatgcga tatttgccga cttaaaaagc 3721 tcaagtgctc caaagaaaaa ccgaagtgcg ccaagtgtct gaagaacaac tgggagtgtc 3781 gctactctcc caaaaccaaa aggtctccgc tgactagggc acatctgaca gaagtggaat 3841 caaggctaga aagactggaa cagctatttc tactgatttt tcctcgagaa gaccttgaca 3901 tgattttgaa aatggattct ttacaggata taaaagcatt gttaacagga ttatttgtac 3961 aagataatgt gaataaagat gccgtcacag atagattggc ttcagtggag actgatatgc 4021 ctctaacatt gagacagcat agaataagtg cgacatcatc atcggaagag agtagtaaca 4081 aaggtcaaag acagttgact gtatcggcca attttaatca aagtgggaat attgctgata 4141 gctcattgtc cttcactttc actaacagta gcaacggtcc gaacctcata acaactcaaa 4201 caaattctca agcgctttca caaccaattg cctcctctaa cgttcatgat aacttcatga 4261 ataatgaaat cacggctagt aaaattgatg atggtaataa ttcaaaacca ctgtcacctg 4321 gttggacgga ccaaactgcg tataacgcgt ttggaatcac tacagggatg tttaatacca 4381 ctacaatgga tgatgtatat aactatctat tcgatgatga agatacccca ccaaacccaa 4441 aaaaagaggg caagccgatc cctaaccctc tgctgggcct ggacagcact ggaagcggag 4501 ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctgaat 4561 tcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 4621 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 4681 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 4741 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 4801 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 4861 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 4921 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 4981 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 5041 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 5101 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 5161 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 5221 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt 5281 agtaagaatt cctcgagcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 5341 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 5401 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 5461 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 5521 tgctggggag gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg 5581 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga 5641 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg 5701 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa 5761 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 5821 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt 5881 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 5941 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt 6001 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 6061 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt 6121 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt 6181 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc 6241 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 6301 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 6361 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 6421 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 6481 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 6541 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 6601 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 6661 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 6721 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 6781 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 6841 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 6901 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 6961 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 7021 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 7081 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 7141 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 7201 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 7261 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 7321 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 7381 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 7441 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 7501 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 7561 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 7621 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 7681 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 7741 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 7801 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 7861 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 7921 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 7981 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 8041 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 8101 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 8161 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 8221 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 8281 cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg 8341 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg 8401 gccaactcca tcactagggg ttcctgcggc cgcacgcgtt gtacatagta atcaattacg 8461 gggtcattag ttcatagccc atatatggag ttccg //