LOCUS Exported 7307 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION FRET biosensor for studying SYK activities in cells. ACCESSION . VERSION . KEYWORDS Cytosolic Syk Biosensor SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7307) AUTHORS Xiang X, Sun J, Wu J, He HT, Wang Y, Zhu C TITLE A FRET-Based Biosensor for Imaging SYK Activities in Living Cells. JOURNAL Cell Mol Bioeng. 2011 Dec;4(4):670-677. doi: 10.1007/s12195-011-0211-x. PUBMED 25541586 REFERENCE 2 (bases 1 to 7307) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7307 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 629..648 /label=T7 /note="T7 promoter, forward primer" promoter 629..647 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 667..676 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 688..705 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 709..741 /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10" /label=T7 tag (gene 10 leader) /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS 745..768 /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /label=Xpress(TM) tag /translation="DLYDDDDK" primer_bind complement(820..841) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1081..1100) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1428..1449 /label=EGFP-C /note="EGFP, forward primer" CDS 1849..2559 /codon_start=1 /product="yellow fluorescent protein optimized for FRET with CyPet (Nguyen and Daugherty, 2005)" /label=YPet /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL LCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLEF LTAAGITEGMNELYK" promoter complement(2626..2644) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2627..2644) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2664..2681) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2670..2894 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2940..3368 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3027..3046) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3237..3258 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3377..3397) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3382..3711 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3562..3697 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3624..3643 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3778..4572 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3832..3851) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4442..4461 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4746..4867 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4783..4802) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4837..4856 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4916..4932) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4916..4932) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4929..4951) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4940..4956 /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(4964..4994) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5009..5030 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5147..5164) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5318..5906) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5398..5417) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6077..6937) /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 6700..6719 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6938..7042) /gene="bla" /label=AmpR promoter primer_bind complement(7117..7136) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt 661 gcggccgcca ccatggtgcg gggttctcat catcatcatc atcatggtat ggctagcatg 721 actggtggac agcaaatggg tcgggatctg tacgacgatg acgataagga tcccatggtg 781 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac 841 gtaaacggcc acaggttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag 901 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg 961 accaccctga cctggggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac 1021 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgtaccat cttcttcaag 1081 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac 1141 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg 1201 gagtacaact acatcagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc 1261 aaggcccact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac 1321 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 1381 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg 1441 gagttcgtga ccgccgcccg catgcattgg tattttggga agatcactcg tcgggagtcc 1501 gagcggctgc tgctcaaccc cgaaaacccc cggggaacct tcttggtccg ggagagcgag 1561 acgacaaaag gtgcctattg cctctccgtt tctgactttg acaacgccaa ggggctcaat 1621 gtgaagcact acaagatccg caagctggac agcggcggct tctacatcac ctcacgcaca 1681 cagttcagca gcctgcagca gctggtggcc tactactcca aacatgctga tggcttgtgc 1741 caccgcctga ctaacgtctg tgggtctaca tctggatctg ggaagccggg ttctggtgag 1801 ggttctggcg atgatatcta cgaagacatc atcaaagtgg aagagctctc taaaggtgaa 1861 gaattattca ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac 1921 aaattttctg tctccggtga aggtgaaggt gatgctacgt acggtaaatt gaccttaaaa 1981 ttactctgta ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaggt 2041 tatggtgttc aatgttttgc tagataccca gatcatatga aacaacatga ctttttcaag 2101 tctgccatgc cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac 2161 tacaagacca gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta 2221 aaaggtattg attttaaaga agatggtaac attttaggtc acaaattgga atacaactat 2281 aactctcaca atgtttacat cactgctgac aaacaaaaga atggtatcaa agctaacttc 2341 aaaattagac acaacattga agatggtggt gttcaattag ctgaccatta tcaacaaaat 2401 actccaattg gtgatggtcc agtcttgtta ccagacaacc attacttatc ctatcaatct 2461 gccttattca aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttttgact 2521 gctgctggta ttaccgaggg tatgaatgaa ttgtacaaat agtaagaatt ctgcagatat 2581 ccatcacact ggcggccgct cgagcatgca tctagagggc cctattctat agtgtcacct 2641 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt 2701 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta 2761 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 2821 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc 2881 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca 2941 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 3001 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 3061 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 3121 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 3181 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 3241 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata 3301 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 3361 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca 3421 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 3481 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 3541 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 3601 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag 3661 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg 3721 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg 3781 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 3841 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 3901 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 3961 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 4021 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 4081 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 4141 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 4201 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 4261 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 4321 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 4381 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 4441 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 4501 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 4561 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 4621 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 4681 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 4741 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 4801 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 4861 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat 4921 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 4981 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 5041 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 5101 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 5161 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 5221 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 5281 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 5341 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 5401 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 5461 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 5521 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 5581 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 5641 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 5701 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 5761 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 5821 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 5881 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 5941 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 6001 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 6061 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 6121 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 6181 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 6241 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 6301 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 6361 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 6421 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 6481 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 6541 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 6601 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 6661 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 6721 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 6781 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 6841 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 6901 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 6961 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 7021 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat 7081 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata 7141 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa 7201 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt 7261 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgtt //