LOCUS Exported 8872 bp ds-DNA circular SYN 27-MAY-2021 DEFINITION Mammalian expression of p85iSH2 fused to mNeonGreen-eDHFR(69K6). ACCESSION . VERSION . KEYWORDS pPBpuro-mNG-eDHFR(69K6)-p85iSH2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8872) AUTHORS Hatano Y, Suzuki S, Nakamura A, Yoshii t, Atsuta-Tsunoda K, Aoki K, Tsukiji S TITLE A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation JOURNAL bioRxiv 2021.03.16.435568 REFERENCE 2 (bases 1 to 8872) AUTHORS . TITLE Direct Submission JOURNAL Exported May 27, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8872 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(3..458) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(140..161) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 352..371 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 585..607 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 599..616 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" repeat_region 674..708 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" enhancer 1011..1390 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1393..1668 /label=chicken beta-actin promoter intron 1669..2677 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 2601..2623 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" regulatory 2750..2759 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2756..3463 /codon_start=1 /product="bright monomeric yellow-green fluorescent protein derived from LanYFP (Shaner et al., 2013)" /label=mNeonGreen /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ KAFTDVMGMDELYK" regulatory 3011..3020 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind complement(4923..4940) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 5150..5169 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5330..5929 /codon_start=1 /gene="pac from Streptomyces" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA" primer_bind complement(5330..5349) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5826..5846 /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind complement(5956..5973) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5962..6186 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6233..6249) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 6257..6273 /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(6281..6311) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6326..6347 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." repeat_region 6581..6643 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(6684..6704) /label=T3 /note="T3 promoter, forward primer" promoter complement(6684..6702) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(6723..6739) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 6747..6763 /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(6771..6801) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6816..6837 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6954..6971) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7125..7713) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7205..7224) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7884..8744) /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 8507..8526 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8745..8849) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc 661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat 721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg 781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact 841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta 901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat 961 atattttctt gttatagata tcaactagaa tgctagcatg ggcccatctc gacattgatt 1021 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga 1081 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 1141 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 1201 acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 1261 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 1321 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 1381 tattaccatg ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct 1441 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg 1501 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg 1561 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg 1621 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg 1681 ttgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc gcccgccccg gctctgactg 1741 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1801 cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct taaagggctc 1861 cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt 1921 ggggagcgcc gcgtgcggcc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg 1981 gggctttgtg cgctccgcgt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt 2041 gcgggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc 2101 agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct ccccgagttg 2161 ctgagcacgg cccggcttcg ggtgcggggc tccgtgcggg gcgtggcgcg gggctcgccg 2221 tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg 2281 gggagggctc gggggagggg cgcggcggcc ccggagcgcc ggcggctgtc gaggcgcggc 2341 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc 2401 ccaaatctgg cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg 2461 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc 2521 gccgccgtcc ccttctccat ctccagcctc ggggctgccg cagggggacg gctgccttcg 2581 ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc 2641 tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttgt 2701 tgtgctgtct catcattttg gcaaagaatt gggatcctca cgtcaattgg ccaccatggt 2761 gagcaagggc gaggaggata acatggcctc tctcccagcg acacatgagt tacacatctt 2821 tggctccatc aacggtgtgg actttgacat ggtgggtcag ggcaccggca atccaaatga 2881 tggttatgag gagttaaacc tgaagtccac caagggtgac ctccagttct ccccctggat 2941 tctggtccct catatcgggt atggcttcca tcagtacctg ccctaccctg acgggatgtc 3001 gcctttccag gccgccatgg tagatggctc cggctaccaa gtccatcgca caatgcagtt 3061 tgaagatggt gcctccctta ctgttaacta ccgctacacc tacgagggaa gccacatcaa 3121 aggagaggcc caggtgaagg ggactggttt ccctgctgac ggtcctgtga tgaccaactc 3181 gctgaccgct gcggactggt gcaggtcgaa gaagacttac cccaacgaca aaaccatcat 3241 cagtaccttt aagtggagtt acaccactgg aaatggcaag cgctaccgga gcactgcgcg 3301 gaccacctac acctttgcca agccaatggc ggctaactat ctgaagaacc agccgatgta 3361 cgtgttccgt aagacggagc tcaagcactc caagaccgag ctcaacttca aggagtggca 3421 aaaggccttt accgatgtga tgggcatgga cgagctctac aagtctgggt ccgcagccag 3481 tagcgccgga agtatgatca gtctgattgc ggcgttagcg gtagatcgcg ttatcggcat 3541 ggaaaacgcc atgccgtgga acctgcctgc cgatctcgcc tggtttaaac gcaacacctt 3601 aaataaaccc gtgattatgg gccgccatac ctgggaatca atcggtcgtc cgttgccagg 3661 acgcaaaaat attatcctca gcagtcaacc gggtacggac aaaaaaaaga aaaagaaaga 3721 tcgcgtaacg tgggtgaagt cggtggatga agccatcgcg gcgtgtggtg acgtaccaga 3781 aatcatggtg attggcggcg gtcgcgttta tgaacagttc ttgccaaaag cgcaaaaact 3841 gtatctgacg catatcgacg cagaagtgga aggcgacacc catttcccgg attacgagcc 3901 ggatgactgg gaatcggtat tcagcgaatt ccacgatgct gatgcgcaga actctcacag 3961 ctattgcttt gagattctgg agcggcggct gtacaagtcc ggactcagat ctcgacaagg 4021 tagtggtgct ggctctggtg ctggtagtgg cgctggttcc ggtgctggct ctggcgcgcc 4081 tcgagcatcc aagtaccaac aagaccaggt ggtgaaggag gacagcgtag aggctgtggg 4141 cgcccagctc aaggtctacc accagcagta ccaggacaag agccgcgaat atgaccagct 4201 gtatgaagaa tacacacgga cctcccagga gctgcagatg aagcgcacag ccatagaggc 4261 cttcaacgag accatcaaga tcttcgaaga gcagggccag acacaggaga agtgcagcaa 4321 ggagtatttg gagcgcttcc ggcgagaggg aaatgagaag gagatgcaga ggatcctgct 4381 gaactccgag cgactcaagt ctcgcatcgc ggagatacac gaaagccgca cgaagttgga 4441 gcaggatctg cgggcgcagg cctccgacaa ccgtgagatc gacaagcgca tgaacagcct 4501 caaacctgac ctcatgcagc tgcgcaagat cagggaccag tacctcgtgt ggctcaccca 4561 gaaaggtgcc cgacagagga agatcaacga atggctggga atcaagaacg agactgagga 4621 ccagtattca ctgatggagg atgaggacgc cctcccccac cacgaggagc gcacgtgaga 4681 attctgcagt cgacggtacc gcgggcccgg gataagtcaa ctaacttaag ctagcaacgg 4741 tttccctcta gcgggatcaa ttccgccccc cccccctaac gttactggcc gaagccgctt 4801 ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg 4861 caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc 4921 ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga 4981 agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc 5041 tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc 5101 acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc 5161 aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga 5221 tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa acgtctaggc 5281 cccccgaacc acggggacgt ggttttcctt tgaaaaacac gataatacca tgaccgagta 5341 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc 5401 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga 5461 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt 5521 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc 5581 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc 5641 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt 5701 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt 5761 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc 5821 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgagtg 5881 cccgaaggac cgcgcgacct ggtgcatgac ccgcaagccc ggtgcctgat cgatgatcta 5941 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 6001 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 6061 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 6121 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 6181 ctatggcttc tgaggcggaa agaacctgca gcccaagctt ggcgtaatca tggtcatagc 6241 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 6301 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 6361 cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatcttaaaa gttttgttac 6421 tttatagaag aaattttgag tttttgtttt tttttaataa ataaataaac ataaataaat 6481 tgtttgttga atttattatt agtatgtaag tgtaaatata ataaaactta atatctattc 6541 aaattaataa ataaacctcg atatacagac cgataaaaca catgcgtcaa ttttacgcat 6601 gattatcttt aacgtacgtc acaatatgat tatctttcta gggttaatct agtatacgcg 6661 tttaattaat ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca 6721 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 6781 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 6841 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 6901 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 6961 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 7021 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 7081 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 7141 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 7201 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 7261 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 7321 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 7381 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 7441 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 7501 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 7561 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 7621 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 7681 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 7741 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 7801 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 7861 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 7921 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 7981 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 8041 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 8101 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 8161 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 8221 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 8281 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 8341 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 8401 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 8461 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 8521 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 8581 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 8641 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 8701 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 8761 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 8821 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac //