LOCUS Exported 5565 bp ds-DNA circular SYN 03-AUG-2022 DEFINITION Protein Expression. ACCESSION . VERSION . KEYWORDS Phat2-hs-AKT1-PH-EGFP-6H SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5565) AUTHORS Shen Z, Belcheva KT, Jelcic M, Hui KL, Katikaneni A, Niethammer P TITLE A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains. JOURNAL Proc Natl Acad Sci U S A. 2022 Jan 4;119(1). pii: 2112390119. doi: 10.1073/pnas.2112390119. PUBMED 34969839 REFERENCE 2 (bases 1 to 5565) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 3, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5565 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 32..892 /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(250..269) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1063..1651 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1552..1571 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1805..1822 /label=L4440 /note="L4440 vector, forward primer" primer_bind 1893..1912 /label=T7 /note="T7 promoter, forward primer" promoter 1893..1911 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1911..1935 /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)." RBS 1948..1970 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" regulatory 2431..2440 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2437..3153 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 2437..3153 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2482..2503) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2743..2762) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3090..3111 /label=EGFP-C /note="EGFP, forward primer" CDS 3160..3177 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" promoter complement(3207..3225) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(3208..3225) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(3283..3301) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3297..3344 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind complement(3580..3601) /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3751..4833) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 4795..4814 /label=LacI-R /note="LacI, reverse primer" promoter complement(4834..4911) /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." primer_bind 5054..5073 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind complement(5227..5246) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5346..5368 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5406..5424) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5492..31 /gene="bla" /label=AmpR promoter ORIGIN 1 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 61 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 121 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 181 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 241 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 301 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 361 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 421 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 481 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 541 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 601 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 661 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 721 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 781 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 841 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 901 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 961 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 1021 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 1081 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 1141 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 1201 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 1261 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 1321 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 1381 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 1441 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 1501 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 1561 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 1621 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 1681 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 1741 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 1801 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 1861 cgcgcgttgg ccgattcatt aatgcagctg attaatacga ctcactatag ggaattgtga 1921 gcggataaca attcctctag aaataatttt gtttaacttt aagaaggaga tatatccatg 1981 agcgacgtgg ctattgtgaa ggagggttgg ctgcacaaac gaggggagta catcaagacc 2041 tggcggccac gctacttcct cctcaagaat gatggcacct tcattggcta caaggagcgg 2101 ccgcaggatg tggaccaacg tgaggctccc ctcaacaact tctctgtggc gcagtgccag 2161 ctgatgaaga cggagcggcc ccggcccaac accttcatca tccgctgcct gcagtggacc 2221 actgtcatcg aacgcacctt ccatgtggag actcctgagg agcgggagga gtggacaacc 2281 gccatccaga ctgtggctga cggcctcaag aagcaggagg aggaggagat ggacttccgg 2341 tcgggctcac ccagtgacaa ctcaggggct gaagagatgg aggtgtccct ggccaagccc 2401 aagcaccgcg tgaccatgaa cggaccggtc gccaccatgg tgagcaaggg cgaggagctg 2461 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 2521 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 2581 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 2641 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 2701 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 2761 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 2821 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 2881 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 2941 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 3001 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 3061 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 3121 gggatcactc tcggcatgga cgagctgtac aagtccggac atcatcatca tcatcactga 3181 gcggccgcat gcataagctt gagtattcta tagtgtcacc taaatcccag cttgatccgg 3241 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag 3301 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta 3361 tatccggata acctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 3421 gcgcagcctg aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta 3481 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 3541 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 3601 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 3661 ccacagcttg catgcctgca ggtcggaagc ataaagtgta aagcctgggg tgcctaatga 3721 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 3781 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 3841 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc 3901 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc 3961 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc 4021 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc 4081 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat 4141 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg 4201 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac 4261 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc 4321 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc 4381 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc 4441 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg 4501 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc 4561 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc 4621 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac 4681 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc 4741 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata 4801 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc 4861 cgggcgctat catgccatac cgcgaaaggt tttgcaccat tcgatggtgt cgggtaccga 4921 gctcgaattg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac 4981 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg 5041 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag 5101 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt 5161 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 5221 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc 5281 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 5341 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg 5401 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 5461 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 5521 aatacattca aatatgtatc cgctcatgag acaataaccc tgata //